Sulfonamide antimicrobials comprise a large class of synthetic, primarily bacteriostatic agents finding both human and veterinary use. Sulfonamides enter soils and subsurface environments primarily via the disposal of human and animal wastes. Following their release, sulfonamides may undergo several transformation processes including chemical incorporation (i.e., covalent cross-coupling) into natural organic matter. We previously demonstrated the phenoloxidase-mediated cross-coupling of sulfamethazine (SMZ) with model humic constituents (Bialk et al., Environ. Sci. Technol. 2005, in press). Here, we report the oxidative cross-coupling of SMZ with soil humic acids. We incubated [phenyl-¹³C₆]-sulfamethazine with Elliot humic acid in the presence of Arthromyces ramosus peroxidase (ARP). After incubation, samples were freeze dried, dissolved in DMSO-d₆ and examined by nuclear magnetic resonance (NMR) spectroscopy. Gradient-selected heteronuclear single-quantum correlation experiments provided direct spectroscopic evidence of ARP-mediated covalent cross-coupling of sulfonamide antimicrobials with humic substances. The results of additional NMR experiments conducted to determine the nature of the covalent linkages will be discussed. A stable covalent linkage formed via the anilinic nitrogen common to all sulfonamides would be expected to eliminate the bioactivity of these compounds following binding. Bacterial assays were conducted to evaluate the antimicrobial activity of cross-coupled products. Based on the results of these experiments, immobilization and inactivation of sulfonamide antimicrobials by chemical incorporation into natural organic matter should be considered in evaluating their environmental fate.