The present investigation was designed to study the effect of synthetic ACTH on the synthesis of progesterone, 20 alpha-OH-progesterone and adenosine-3', 5'-monophosphate (cAMP) in isolated luteal cells. Experiments were conducted on rats from three groups. Group I: PMS primed immature rats. Group II: PMS plus hCG primed immature rats. Group III: pregnant rats. Luteal cells were isolated by the Kumai Method (7) using a sucrose density gradient after digestion with trypsin and collagenase. Luteal cells were divided into three fractions; S-1, S-2 and S-3. Following an 18 hr-incubation of S-1 cells of day 7 after PMS injection, a significant increase in progesterone release by ACTH lasted from 6 to 18 hr, whereas 20 alpha-OH-progesterone decreased significantly by ACTH from 8 to 18 hr. There were no differences in progesterone and 20 alpha-OH-progesterone releases from S-2 cells of day 7 between media with and without ACTH. Progesterone release without ACTH reached the peak on day 7 in Group I and on day 6 in Group II. ACTH stimulated progesterone and inhibited 20 alpha-OH-progesterone releases in Groups I and II. The maximum effect of ACTH in both Groups was noted on days 6 and 7. There were no differences in the Effective Dose (ED50) and the Inhibitory Dose (ID50) of ACTH between Group I and Group II. In Group III, releases of progesterone and 20 alpha-OH-progesterone from S-1 cells to media with or without ACTH decreased remarkably after 11 days of gestation. On days, 3, 5 and 8 of gestation, a significant increase in progesterone release from S-1 cells was noted by the ACTH addition. 20 alpha-OH-progesterone release from S-1 cells to media with or without ACTH decreased as the pregnancy advanced. Total cAMP of intra- and extra-cellular S-1 cells (Group II) on day 5 after PMS injection was assayed. ACTH induced an increase in intracellular cAMP. The present results indicate the following: (1) In Group I and Group II, progesterone release increased with the age of the luteal cells and ACTH dose. In Group III, ACTH stimulated progesterone release in a dose-dependent manner in early gestation. (2) 20 alpha-OH-progesterone release decreased by ACTH dose in Group I and Group II, and decreased in Group III as the gestation advanced. (3) Intracellular cAMP increased by ACTH in Group II.