The Delta(1)-dehydrogenation of 6-alpha-methyl-hydrocortisone-21-acetate in an organic-aqueous two-liquid-phase system using free Arthrobacter simplex cells was studied. Chloroform, toluene, and n-octan-1-ol were assayed for steroid solubility and biocompatibility, the latter emerging as the most appropriate solvent. Addition of surfactants (a commercial-grade lecithin, phosphatidylcholine, and phosphatidyl-ethanolamine) led to increased initial reaction rates but did not significantly change the final conversion yields. First-order apparent kinetics was observed in a menadione-saturated system for global substrate concentrations under 10 mM. Maintenance stability was considerably increased when the temperature was lowered from 30 to 25 degrees C. However, operational stability levels were unsufficient for biocatalyst reuse in the rested reactional conditions.