Environmental releases and fate of steroid sex hormones from livestock and wastewater treatment plants are of increasing regulatory concern. Despite the detection of these hormones in manures, biosolids, and the environment, little attention has been paid to characterization of fecal bacteria capable of hormone degradation. The enrichments of (swine) manure-borne bacteria capable of aerobic testosterone degradation were prepared and the testosterone mineralization pathway was elucidated. Six DNA sequences of bacteria from the Proteobacteria phylum distributed among the genera Acinetobacter, Brevundimonas, Comamonas, Sphingomonas, Stenotrophomonas, and Rhodobacter were identified in a testosterone-degrading enriched culture with testosterone as the sole carbon source. Three degradation products of testosterone were identified as androstenedione, androstadienedione, and dehydrotestosterone using commercially available reference standards, liquid chromatography-UV diode array detection, and liquid chromatography-time-of-flight mass spectrometry (LC-TOF/MS). Three additional degradation products of testosterone were tentatively identified as 9α-hydroxytestosterone, 9α-hydroxyandrostadienedione or 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, and 9α-hydroxydehydrotestosterone or 9α-hydroxyandrostenedione using LC-TOF/MS. When (14)C-testosterone was introduced to the enriched culture, 49-68% of the added (14)C-testosterone was mineralized to (14)CO(2) within 8 days of incubation. The mineralization of (14)C-testosterone followed pseudo-first-order reaction kinetics in the enriched culture with half-lives (t(1/2)) of 10-143 h. This work suggests that Proteobacteria play an important environmental role in degradation of steroid sex hormones and that androgens have the potential to be mineralized during aerobic manure treatment or after land application to agricultural fields by manure-borne bacteria.