Abstract: The whole cells of Acremonium strictum transformed prednisolone at its side chain to produce two steroid compounds. 21,21-Dimethoxy-11β-hydroxypregn-1,4-dien-3,20-dione was the main metabolite which its production has not been previously reported using microbiological means. This metabolite together with a hydroxylated derivative, 11β-hydroxyandrost-1,4-dien-3,17-dione, were purified with preparative TLC followed by their identification through 1H, 13C NMR and other spectroscopic data. Best fermentation condition was found to be 5 day incubation at 25 °C and pH value of 6 according to TLC profiles. Optimum concentration of the substrate, which gave maximum bioconversion efficiency, was 1 mg mL-1 in one batch. Biotransformation was completely inhibited in a concentration above 5 mg mL-1.