Because of their central role in follicular development, maturation of the ovum, and development of the subsequent corpus luteum, the characterization of granulosa cell function and differentiation is of interest to reproductive biologists. Our objective was to evaluate progesterone (P₄) production by all transretinoic acid (RA, 10 M) alone or in combination with ovine FSH (oFSH, .05 g/ml) in the presence of 22-R hydroxycholesterol (22-R, 1 g/ml) as a substrate by transformed rat granulosa cells (DC-3) in culture over a period of 96h. Cells (~50,000/well) were cultured in Iscove's Modified Dulbecco's Medium (2% fetal bovine serum) in wells in a total volume of 3 ml. Culture medium was collected once every 24h following addition of RA and/or oFSH for up to 96h. Experiments were conducted in triplicate and medium was quantitated for P₄ by RIA. In the presence of substrate alone all treatments produced significant amounts of P₄ daily, compared to respective controls. Daily production of P₄ with 22-R alone ranged between .023 to .046 ng/ml. Addition of oFSH did not affect this secretion rate and P₄ ranged between .025 and .045 ng/ml. However, addition of RA alone increased P₄ production to .21 ng/ml (24h), .89 ng/ml (48h), .71 ng/ml (72h), and declined thereafter to .13 ng/ml (96h). Addition of oFSH in combination with RA gave similar responses during the same time perods, .23 ng/ml (24h), .96 ng/ml (48h), .76 ng/ml (72h) and .31 ng/ml (96h), respectively.