The objective of this study was to investigate the direct effect of estradiol-17 beta (E2) and progesterone (P4) on LH release by pituitary cells of midluteal phase cows in vitro. Pituitaries were collected at the slaughterhouse; cells were dissociated with collagenase and hyaluronidase and maintained in a static culture system in Medium 199 (M199). Various concentrations of E2 (0.1-100 nM) and P4 (0.1 and 10 nM) were used to stimulate the cells for 2, 4, 6, 15, 24, 48, or 72 h. In addition, the synergistic action of E2 and P4 was investigated by exposure of the cells to a combination of the two hormones. At the end of each incubation, the cells were challenged with LHRH (1 nM) for 2 h. The medium was collected for LH analysis at the end of each incubation period and after the LHRH challenge; furthermore, intracellular LH content was quantified at the end of each experiment. The results indicate a positive action of E2 on basal release of LH beginning after 15 h of exposure (p < 0.01). LHRH-induced LH release was modulated by E2 in a time-dependent manner with an effect at first inhibitory, then stimulatory, and finally inhibitory again (p < 0.04). P4 alone did not affect LH release, but it negatively influenced LHRH-induced LH release. P4 also exerted a positive action on intracellular LH after 6 h of incubation. A substantial inhibitory effect (p < 0.001) on both tonic LH release and LHRH-induced LH release was observed in cells exposed for 16 h to P4 after a priming with E2 for 4 h.(ABSTRACT TRUNCATED AT 250 WORDS)