The bioconversion of cortexolone (Reichstein's Compound S) to hydrocortisone or hydrocortisone to prednisolone by reference and isolated fungi and bacteria were investigated, The reference strains Cunninghamella echinulata ATCC 8688a and Bacillus sphaericus ATCC 13805, and the isolates C echinulata SRP III and B sphaericus SRP III were used as free or immobilized farms in single or mixed cultures by either sequential or concomitant mixing. In single cultures, immobilized fungi C echinulata ATCC 8688a and C echinulata SRP III showed less hydrocortisone yield than the free form, whereas immobilized B sphaericus ATCC 13805 and B sphaericus SRP III gave higher prednisolone yield than the free form. In mixed cultures, free fungi with immobilized bacteria of both isolated and standard strains showed the highest prednisolone yield both by concomitant and sequential mixing. The highest prednisolone yield of 72.5 +/- 0.78% with a bioconversion rate of 0.76% h(-1) by sequential mixing was obtained with free C echinulata ATCC 8688a with immobilized B sphaericus ATCC 13805 at 96 h, whereas the free C echinulata SRP III with immobilized B sphaericus ATCC 13805 and the free C echinulata SRP III with immobilized B sphaericus SRP III gave 63.4 +/- 0.74% and 47.7 +/- 0.44% at 120 h with bioconversion rates of 0.53 and 0.40% h(-1) respectively. Free C echinulata SRP III with immobilized B sphaericus SRP III gave a maximum yield of 52.8 +/- 0.82% with a bioconversion rate of 0.44% h(-1) at 120 h which was comparable to 59.2 +/- 0.44% with a bioconversion rate of 0.49% h(-1) obtained by free C echinulata ATCC 8688a with immobilized B sphaericus ATCC 13805 using concomitant mixing. This study indicated high biotransformation activity of free fungal and immobilized bacterial isolates which showed comparable steroidal productivity to the standard strains both in single and mixed cultures by the concomitant mixing technique.