Using a preparation of highly purified, adult rat Leydig cells and conditions of culture which was found to optimize testosterone production during 24 h, attempts were made to maintain optimal testosterone production for 3 d. Leydig cells cultured on Cytodex 3 beads at 19% O2 in Dulbecco's modified Eagle's medium-Ham's nutrient mixture F12 (1:1; vol/vol) containing 0.5 mg/ml total bovine lipoproteins (<1.222g/ml) with maximal luteinizing hormone (LH) stimulation failed to maintain a constant amount of testosterone for 3 d. These cells did however secrete a similar amount of total delta 4-3-ketosteroids on each of the 3 culture d, indicating that their viability was preserved. The predominance of progesterone and 170H-progesterone relative to the amount of androstenedione found on Days 2 and 3 suggested that the activity of the cytochrome P450C17-hydroxylase-C17,20-lyase enzyme in the smooth endoplasmic reticulum was diminished when Leydig cells were maintained in our primary culture for longer than 24 h. Decreasing the oxygen tension of the cultures from 19 to 5%, and decreasing the concentration of LH used to stimulate the Leydig cells from 100 to 0.1 ng/ml, were necessary to achieve maintenance of testosterone secretion without accumulation of other delta 4-3-ketosteroids during a 3-d period. Cells cultured in this fashion were still able to respond to maximal LH stimulation during Day 3, producing as much testosterone as if cultured for 24 h on Day 1 at 19% O2 with 100 ng/ml LH stimulation. (Copyright (c) 1989 Tissue Culture Association, Inc.)