Summary-High molecular weight, linear polyacrylamide (PAM) with anionic charge is added to agricultural soils as an anti-erosion additive. Research indicates that soil microorganisms are able to utilize PAM as a source of N and that inorganic N pools are altered in some PAM-treated soils. The potential role of hydrdlytic amidase activity in the microbial utilization of PAM for N was investigated. Intracellular and extracellular amidase activity was measured over time in enrichment cultures which used PAM as sole N source. Enzyme activity increased concomitant with cell growth and N removal from PAM. Cell growth, N removal and amidase production were dependent upon readily-available C in the medium. Amidase activity and substrate specificity were determined for PAM-utilizing enrichment cultures exposed to various N sources. Polyacrylamide-specific amidase activity appears to be inducible, and not constitutive, based on the lack of amidase activity in cultures supplied with only ammonium nitrate for N versus substantial activity when PAM was added as an amendment with or without ammonium nitrate. Cultures amended with propionamide exhibited amidase activity largely specific for this small amide substrate, while cultures supplied with PAM as sole N source exhibited amidase activity specific for formamide, propionamide and PAM. Amidase activity and substrate specificity were determined for PAM-treated and untreated agricultural field soils. Polyacrylamide-specific amidase activity was higher in PAM-treated soil (14.86 ± 14.0 pg NH4 released soil) than in untreated soil (1.02 ± 2.3 pg NH4 released C I soil); activity specific for low molecular weight amides was slightly elevated or unchanged in PAM-treated soil as compared with untreated soil.