Luteal cells from the corpora lutea of rats on day 15 of pregnancy were suspended in DMEM-F12 and progesterone accumulation was measured by radioimmunoassay. Progesterone accumulation increased steadily over eight hours and then gradually decreased. Progesterone accumulation was seen to increase with the addition of BSA and 25-hydroxycholesterol. At doses of 12 ng/ml and 600 ng/ml hCG, progesterone accumulation increased, but was seen to decrease at a dose of 30 micrograms/ml hCG. However, the addition of cyclohexamide alone had no effect on progesterone accumulation, but the simultaneous addition of cyclohexamide and hCG blocked the effects of hCG in a dose related manner. These data indicate that progesterone production by rat luteal cells depends on cholesterol availability. The results reveal that hCG stimulates progesterone production by a process requiring new protein synthesis.