The effects of preincubating rat granulosa cells with porcine LH, and its dissociated alpha and beta subunits on subsequent Prl mediated progesterone secretion were examined. Granulosa cells were isolated from ovarian follicles 50 h after injection of 5 IU PMSG and were then cultured as monolayers in serum supplemented medium. Cells were preincubated for 24 h with medium alone (control) or with 0.1 or 1 microgram/ml of LH, LH alpha, LH beta, or both LH alpha and LH beta. Cells were also preincubated with 50 ng/ml LH alpha and 50 ng/ml LH alpha that had been further purified with immunoaffinity chromatography. Following the preincubation period, cells were maintained an additional 6 days in the presence or absence of 1 microgram/ml Prl. Preincubation with LH or its individual subunits markedly increased the responsiveness to Prl stimulation through 6 days of culture, whereas cells cultured in the absence of Prl secreted decreasing amounts of progesterone. Preincubation with the dissociated subunits increased Prl mediated progesterone secretion approximately two-thirds as much as preincubation with intact pLH. Immunopurified pLH alpha increased the responsiveness to Prl to the same degree as non-purified pLH alpha, suggesting that the stimulatory effects of the subunits are not due to contamination by intact LH. The magnitude of Prl mediated progesterone secretion was dependent upon the preincubation concentration of LH or its dissociated subunits in most, but not all treatments. The increased responsiveness to Prl suggests that in the proper endocrine environment, individual subunits of gonadotrophins may functionally interact with target cells.