Oxygen deficiency caused by a decrease in the blood supply is known to induce various responses of cells. Since luteal blood flow has been shown to decrease during luteolysis, a low oxygen condition seems to be an integral part of the environment during luteolysis. To determine whether a low oxygen condition is associated with functional luteolysis, we examined the influence of reduced oxygen tension on the luteal progesterone (P4) generating system in cultured bovine mid luteal cells. Luteal cells obtained from mid-cycle corpus luteum (Days 8-12) were incubated under different O₂ concentrations (20%, 10%, 5%, 3% O₂) with or without luteinizing hormone (LH) for 24 h. P4 production decreased with decreasing O₂ concentration but was significantly stimulated by LH regardless of O₂ concentration. After 8 h of culture, both basal and LH-stimulated P4 production were significantly lower under 3% O₂ than under 20% O₂. Low oxygen condition also inhibited pregnenolone production. Cytochrome P450 side-chain cleavage enzyme (P450scc) mRNA expression, measured by quantitative PCR, decreased under low oxygen condition in both non-LH-treated and LH-treated cells. Low oxygen condition did not affect the expressions of steroidogenic acute regulatory protein (StAR) mRNA or protein, while StAR mRNA expression was stimulated by LH during 4 h of culture. Low oxygen condition also did not affect 3-hydroxysteroid dehydrogenase/5-4 isomerase mRNA expression or the activity of the enzyme in the cells regardless of the incubation period. The overall results indicate that a low oxygen condition decreases P4 synthesis by attenuating P450scc production and P450scc activity in bovine luteal cells, and suggest that oxygen deficiency is an essential condition for the progression of luteolysis in cattle.