Technetium-99 is a radioactive contaminant in marine and terrestrial environments. Under oxic conditions, it is present as the pertechnetate anion [Tc(VII)O₄^-], which is soluble and mobile in the environment. In paddy fields in Japan, the accumulation of Tc in the 20-cm plow layer was observed (Tagami and Uchida 2002). Since paddy fields are flooded during the planting season, reductive conditions develop in the plowed layer. Under reductive conditions, Tc is transformed from Tc(VII)O₄^- to Tc(IV) species. Tc(IV) species generally are insoluble and immobile in the environment, and thus Tc(IV) could accumulate in the plowed layer. Recently, it was found that gram-negative-bacteria were responsible for the formation of insoluble Tc in the ponding water of flooded paddy fields (Ishii and Uchida 2005). This insolubilization would be one of the factors of the Tc accumulation in the plowed layer because bacteria sink into the plowed layers due to their own weight over time. However, little information is available for environmental conditions of Tc insolubilizing by the bacteria. In this study, optimum oxic conditions for the insolubilization were determined. Ponding water samples were collected from paddy soil which had been flooded for 7 days. As a redox indicator, the concentration of Fe²⁺ in the samples was measured immediately upon sample collection using a 1, 10-phenanthroline monohydrate method. The ponding water samples were amended with a NH₄⁹⁹TcO₄ spike and incubated aerobically and anaerobically for 7 days at 25°C in the dark. In control experiments, bacterial cells were killed by the addition of a broad-spectrum beta-lactam antibiotic, chloramphenicol. After the incubation, the samples were filtered through 0.2-µm pores and the radioactivity of ⁹⁹Tc in the obtained filtrates was measured with Tri-Carb-25WTR Liquid Scintillation Analyzer. The relative amount of insoluble Tc was calculated from the total radioactivity in the sample and the radioactivity in the filtrate samples. Different valences of Tc were separated on Merck TLC plastic sheets cellulose with 0.3 N HCl as the mobile phase and assayed by autoradiography with a FLA-5100 imaging system (Fuji Film, Japan). The concentration of Fe²⁺ in the ponding water samples was 183.0 ± 8.8 µg mL^-1 at day 7 of flooding. Fe²⁺ in the samples must have been released from the soil, which established reductive conditions by the flooding because hardly any Fe²⁺ was released from the soil by shaking for 30 min at day 0 of flooding. Valences of Tc were determined by autoradiography after 7 days of incubation under anaerobic conditions. A significant amount of Tc(VII) (Rf of 0.67) was observed, but the other Tc species were negligible. This result suggested little transformation of Tc from Tc(VII)O₄^- to Tc(IV) species in the ponding water samples. Therefore, transformation of Tc under reductive conditions is not enough to explain the accumulation of Tc in the plowed layer. The formation of insoluble Tc by bacteria was determined using the killed control and the untreated ponding water samples. The samples were statically incubated. The relative amount of insoluble Tc in the killed control was 1.8 ± 16.8% of total Tc added, while in the untreated ponding water sample it was 52.7 ± 18.0 %. These results suggested that the formation of insoluble Tc in ponding water samples was mainly caused by bacteria. The ponding water samples spiked with Tc were incubated under aerobic and anaerobic conditions. The relative amounts of insoluble Tc under aerobic and anaerobic conditions were 6.9 ± 3.8 and 61.1 ± 3.0% of total Tc added, respectively. Therefore, it was found that anaerobic conditions promoted more formation of insoluble Tc by bacteria. A series of radioactive tracer experiments showed the formation of insoluble Tc by bacteria. This formation was promoted more under anaerobic conditions compared with aerobic conditions. Even under anaerobic conditions, Tc was present as Tc(VII) in the ponding water samples. To elucidate the mechanism of the formation of insoluble Tc, it is necessary to isolate Tc insolubilizing bacteria from the ponding water samples. This work has been partially supported by the Agency for Natural Resources and Energy, the Ministry of Economy, Trade and Industry (METI) Japan.