Previously, we found that single applications of biosolids have long term (12-plus years) effects on rangeland plant communities in the Colorado shortgrass steppe. Specifically, plant species diversity decreased and communities became increasingly dominated by western wheatgrass, a facultative mycotroph, as biosolids application rate increased from 0 to 30 Mg ha^-1. We also found that relative amounts of the fatty acid methyl ester biomarker for arbuscular mycorrhizal (AM) fungi, 16:1w5c, decreased in soil as biosolids application rate increased. In this study, we made direct measurements of AM fungi and their functions to determine any long-term detrimental impacts of biosolids when land-applied to a rangeland soil. The experimental research site consisted of replicate (4) 7.5 x 15 m plots amended with biosolids (0, 2.5, 5, 10, 20, or 30 Mg ha^-1) in 1991. Soil and plant roots were collected in July 2004. A chromogenic detection-based in situ hybridization assay, using AM1 and Gig15.1 fungal primers, was developed to measure lengths of AM fungal hyphae in soil. Plant-mycorrhizal associations were assessed by measuring the percent of roots colonized by AM fungi for western wheatgrass as well as blue grama, a mycorrhizal host species whose biomass declined with increasing biosolids application rate. We also quantified the concentration of glomalin in plot soils by the total protein Bradford assay. Our results will provide a clearer understanding of the long-term effects of a single biosolids application on plant-microbial interactions, as well as the relationship of the 16:1w5c marker to other measures of AM fungi.