Isolation of progesterone secreting cells from the ovaries of immature rats pretreated with pregnant mare serum gonadotropin (PMS) was conducted by a simple procedure which combined the collagenase digestion with a density gradient method. After digestion of the ovarian tissue slices by the enzyme, the residue was gently pressed and placed on a sucrose density gradient. Three bands appearing in the tube after centrifugation were designated as S-1, S-2 and S-3 from the top to the bottom, respectively. The S-1 cells from the ovaries at 6 days after PMS secreted the greatest amount progesterone, i.e. approximately 430 ng per 10(5) cells during the 18th incubation. Progesterone secretion from the S-2 cells was less than 48% of that from the S-1 cells. A physiological interrelation between the S-1 and S-2 cells remains unexplained by the present experiment. The luteal cells were yellow, spheroidal and 15 to 40 mus in diameter. Many vesicle-like particles were found on the cell surfaces. Progesterone secretion from the prepared cells was stimulated significantly by hCG in vitro. This result indicates that progesterone secreting cells isolated by the collagenase-sucrose density gradient preserve their native function as luteal cells. The procedure for preparation of luteal cells in the present report may provide a suitable model for in vitro studies on the corpus luteum.