Corpora lutea were removed from regularly cycling dairy cows, dissociated with collagenase and cultured for 8 or 10 days in Ham's F-12 medium. In Exp. 1 treatment with insulin, or an insulin-transferrin-selenium combination (ITS), increased progesterone production from basal levels on Day 4 of culture to 234% (P less than 0.01) above controls on Day 10. LH alone increased progesterone production 45% above controls on Day 10 (P greater than 0.05). When LH was combined with insulin or ITS, progesterone production was stimulated to an average of 1802% (P less than 0.01) above controls on Day 10 of culture. Transferrin or selenium without insulin did not allow LH to stimulate progesterone synthesis. In Exp. II, LH alone or LH plus gentamicin or penicillin-streptomycin increased progesterone production from basal levels on Day 2 steadily to an average of 468% (P less than 0.01) above controls (no antibiotics) by Day 8 of culture. The addition of amphotericin-B, alone or in combination with the other antibiotics, inhibited all LH-stimulated progesterone synthesis, but did not affect basal progesterone levels. We conclude that insulin is essential for maximal steroidogenesis in a bovine luteal cell culture system, and that LH-stimulated progesterone production is inhibited in the presence of amphotericin-B, but is not inhibited by gentamicin or penicillin-streptomycin. The elimination of amphotericin-B, coupled with the addition of insulin to the cell culture system increased the responsiveness of the cells to LH. These culture conditions represent the first report in which LH increased total progesterone production for 10 days, maintaining luteal function in a chemically-defined culture system.