Cultured Leydig cells exhibited time-dependent decreases in the microsomal cytochrome P-450 enzyme activities, 17 alpha-hydroxylase and C17-20 lyase when maintained under standard culture conditions (95% air, 5% CO2). Inclusion of the hydroxyl radical scavenger dimethyl sulfoxide in the culture medium, or the reduction of oxygen tension from 19 to 1% O2 was effective in preserving these enzyme activities and the combined effects of low O2 and dimethyl sulfoxide were synergistic. Leydig cells in culture were treated with 1 mM 8-Br-cAMP to induce steroidogenic desensitization which resulted in greater decreases in 17 alpha-hydroxylase and C17-20 lyase activities, as well as a diminished capacity to produce testosterone in response to subsequent acute stimulation with 8-Br-cAMP. Reduction of the oxygen tension from 19 to 1% O2 prevented this enhanced loss of microsomal P-450 activities in desensitized Leydig cells. The activity of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase, a microsomal enzyme which is not a P-450 enzyme, was stable in cultures of both control and desensitized Leydig cells under all culture conditions. These data are consistent with the hypothesis that oxygen-mediated damage is responsible for the time-dependent decrease in 17 alpha-hydroxylase and C17-20 lyase activities of control Leydig cells, and is the mechanism by which these microsomal P-450 activities are further decreased in desensitized Leydig cells. Desensitized Leydig cells exhibited a 50 and 70% decrease at 24 and 48 h, respectively, in their ability to produce testosterone in response to subsequent acute stimulation with 8-Br-cAMP, regardless of the culture conditions. Since desensitized Leydig cells cultured at 1% O2 showed no greater loss of enzyme activity than did controls, loss of microsomal P-450 activities is not the cause of the diminished testosterone biosynthetic capacity of desensitized Leydig cells.