We examined the participation of the intraluteal levels of progesterone (P4) and prostaglandin F2 alpha (PGF2 alpha) in the induction of luteolysis by LH and its relationship with the induction of the 20 alpha-hydroxysteroid dehydrogenase activity (20 alpha-HSD). Subcutaneous administration of four doses of 10 microgram ovine LH (oLH) at 0800, 0900, 1000 and 1100 h on day 19 of pregnancy induced a decrease in the activity of the enzyme 3 beta-HSD 24 and 48 h after treatment and an increase in luteal 20 alpha-HSD activity 48 h after oLH treatment when compared with control rats. Intraluteal and serum P4 levels were lower than control values 24 and 48 h after oLH treatment, with a significant increase in luteal PGF2 alpha content and a decrease in corpus luteum (CL) weight 48 h after oLH treatment. Intrabursal ovarian (i.b.) treatment with an inhibitor of PG's biosynthesis (diclofenac) (70 microgram/ovary) or P4 (3 microgram/ovary) on day 20 of pregnancy, prevented the increase in 20 alpha-HSD activity observed 48 h after oLH treatment, without any effect on 3 beta-HSD activity. The i.b. administration of P4 prevented the increase in intraluteal PGF2 alpha content induced by oLH treatment and the increases in 20 alpha-HSD activity and intraluteal PGF2 alpha content observed in control animals on day 21 of pregnancy. The inhibition of PG biosynthesis also prevents the decrease in intraluteal and serum P4 level induced by oLH. These results provide good evidence of the important participation of intraluteal P4 and PGF2 alpha on the oLH-induced luteolysis in pregnant rats. We also found the P4 produced by the CL is involved, in part, in the regulation of luteal PG synthesis. Thus, the early decline in 3 beta-HSD activity and the consequent fall in intraluteal P4 content, may trigger the synthesis of PGs and thereafter the increase in luteal 20 alpha-HSD activity to establish luteolysis.