Bioconversion of hydrocortisone to prednisolone by free, immobilized and reused immobilized cells of three bacterial strains (Bacillus sphaericus ATCC 13805, Bacillus sphaericus SRP III and Arthrobacter simplex 6946) in an aqueous and a two-liquid-phase system using different organic solvents was investigated. The experiments were carried out in a 125 cm(3) shake flask at 27 +/- 2 degrees C, 220 rpm for 96 h. The contents of prednisolone and hydrocortisone in samples taken at 0, 3, 6, 24, 48, 72, 96 and 144 h were determined by HPLC analysis. The immobilized bacterial cells showed higher prednisolone yield than the free form in an aqueous system. In the two-phase systems, the butyl acetate to aqueous media ratio of 1 :30 for all three bacterial strains in immobilized forms gave the highest prednisolone yields, at an incubation time of 144 h, of 87.6, 70.6 and 88.3% respectively. For an n-decane to aqueous ratio of 1:6, moderate prednisolone yields of 81.8, 47.9 and 71.4% were obtained with shorter incubation times of 72, 96 and 6 h respectively. For cyclohexane and other alcohols, the organisms produced low yields of prednisoione (0-30%). Single reuse of all three immobilized bacterial cells gave a 3-20% lower yield of prednisolone than the non-reused cells. The increase in hydrocortisone concentration decreased the prednisolone production whereas increasing the n-decane to aqueous ratio from 1:6 to 1:3 caused no significant change in the productivity.