The in vitro ability of ovine (o) follicle-stimulating hormone (FSH), (o)luteinizing hormone (LH), (o)prolactin (PRL), and recombinant human FSH (rhFSH) to stimulate progesterone (P4) synthesis by rat corpora lutea on Day 4 of pregnancy was investigated. Dispersed luteal cells (large + small cells) were incubated in the presence of the gonadotropins (1-100 ng) alone or in various combinations (10 ng each) for 4 or 24 hr. Given alone, all the ovine preparations stimulated P4 in a dose-dependent manner with even 1 ng of each hormone significantly enhancing P4 production. Significantly, rhFSH--which is devoid of LH contamination--at 10 and 100 ng also stimulated P4 production, thus clearly establishing for the first time that FSH is a luteotropic hormone in the rat. The combination of oFSH + LH + PRL (10 ng each) significantly stimulated P4 synthesis to a greater extent than the combination of any two hormones or individual hormones at both 4 hr or an additional 24 hr of incubation (P < 0.05). This verified in vitro a previously established in vivo luteotropic complex. One hundred nanamolars of phorbol 12-myristate 13-acetate (PMA) did not affect basal P4 secretion but inhibited cAMP, oFSH, and oLH stimulation of P4. Thus, the luteotropic effects of FSH, LH, and activators of protein kinase A are antagonized by the protein kinase C pathway.