The effects of preincubating rat granulosa cells with FSH, LH, and Prl on subsequent Prl mediated progesterone secretion were investigated. Granulosa cells were isolated from ovarian follicles 50 h after injection of 5 IU PMSG and were then plated on poly-L-lysine coated coverslips in serum supplemented medium. Cells were preincubated for 24 h in the absence of hormones (control) or with the addition of either 0.25, 2.5, 25 ng/ml rat FSH or rat LH, or 1 microgram/ml rat Prl. Following the preincubation period, cells were maintained for an additional 6 or 8 days in the presence or absence of 1 microgram/ml Prl. When cells were preincubated with FSH or LH, only the two higher concentrations (2.5 and 25 ng/ml) stimulated significantly more progesterone secretion than control cultures during the 24 h preincubation period. For each series of preincubations, cells cultured for 6 or 8 days in the presence of Prl secreted significantly more progesterone at each day of culture than cells cultured without Prl. Cells preincubated and cultured with Prl secreted only 3-7-fold more progesterone than cells preincubated in control medium and then cultured with Prl. Preincubation with FSH or LH promoted a 20-45-fold increase in Prl mediated progesterone secretion compared to control preincubation cultures that also subsequently were cultured with Prl. The magnitude of Prl mediated progesterone secretion observed through 6 days of culturing was dose dependent on the preincubation concentration of FSH or LH.(ABSTRACT TRUNCATED AT 250 WORDS)