The present work aimed at quantifying the viability and morphological changes occurring during the time course of the side-chain cleavage of beta-sitosterol, in aqueous, two-phase organic-aqueous and organic media by free resting cells of Mycobacterium sp. NRRL B-3805. The solvent used was bis(2-ethylhexyl) phthalate (BEHP). A 66.3% reduction in cell viability was observed after 24 h when the cells were incubated in phosphate buffer only, but the percentage of viable cells was constant thereafter. In biphasic systems with BEHP, cell viability was maintained at higher values in the first 48 h, during which complete degradation of substrate was achieved. The availability of oxygen, which should be higher in the biphasic system than in the aqueous system, and of a carbon and energy source, thus seem important for the cells to retain their viability. In biphasic systems, cells tended to shrink and decrease their surface roughness, i.e. to decrease their surface area, possibly as a way to protect themselves from mechanical stress due to the presence of organic-aqueous interfacial forces, which resulted in disaggregation of cell clusters. A method used to visualise BEHP droplets with a standard optical microscope showed that the cells adhered to the surface of the solvent droplets, but no cells were observed inside these. In pure BEHP medium, cells retained their viability level for at least 150 h, independently of a pre-incubation period, which did not seem to induce any adaptation effect. Solvent biocompatibility, higher oxygen availability and reduced interfacial stress could have contributed to this maintenance of viability.