Bioconversion of 16-dehydropregnenolone acetate (16-DPA) to androsta-1,4-diene-3,17-dione (ADD), an intermediate for the production of female sex hormones, by mixed culture of Pseudomonas diminuta MTCC 3361 and Comamonas acidovorans MTCC 3362 is reported. Various physicochemical parameters for the bioconversion of 16-DPA to ADD have been optimized in shake flask cultures. Nutrient broth inoculated with actively growing co-culture proved ideal for bacterial growth and bioconversion. A temperature range of 35-40 degreesC was most suitable; higher or lower temperatures adversely affected the bioconversion. Dimethylformamide below 2% concentration was the most suitable carrier solvent. Maximum conversion was recorded at 0.5 mg mL(-1) 16-DPA. A pH of 5.0 yielded a peak conversion of 62 mol % in 120 h incubation period. Addition of 9alpha-hydroxylase inhibitors failed to prevent further breakdown of ADD to nonsteroidal products. 16-DPA conversion in a 5 L fermenter followed a similar trend.