This study investigated the effects of physiological concentrations of GnRH and estradiol (E2) on LH biosynthesis and release using cultured anterior pituitary cells. Pituitaries from female rats were enzymatically dispersed and cultured for 48 h in steroid-free Î±-Modified Eagleâ€™s Medium, followed by a 24-h culture in medium with or without E2. The cells were then incubated for a 4-h (Exp 1 and 2) or 8-h (Exp 3) period in medium containing radiolabeled precursors with or without GnRH. Radioactive precursor incorporation into LH was determined by immunoprecipitation, while immunoreactive LH (iLH) content was quantified by RIA. In the first experiment, all concentrations of E2 (10-11-10-8 M) enhanced iLH release in response to 1 nM GnRH, confirming previous reports. GnRH increased [3H]glucosamine (3H-Gln) incorporation into LH, but had no effect on [35S]methionine (35S-Met) incorporation. The higher concentrations of E2 enhanced GnRH-stimulated 3H-Gln LH production. In the second experiment, the effects of GnRH (10-9 M) and E2 (5 Ã— 10-10 M) on the incorporation of [3H]galactose, [3H]mannose, [3H]fucose, or [35S]sulfate into LH were investigated. Although all precursors were incorporated into LH, no specific effect of GnRH and/or E2 on incorporation of any of the precursors into LH was noted. In Exp 3, pituitary cells were cultured with or without 0.5 nM E2 followed by an 8-h incubation with varying physiological concentrations of GnRH (10-11-10-9 M) and radiolabeled precursors (3H-Gln and 35S-Met). GnRH stimulated iLH release in a dosedependent manner, and this response was enhanced by E2. GnRH also increased the incorporation of both 3H-Gln and 35SMet into LH, but the dose of GnRH required for this response was dependent upon the estrogen environment. In the absence of E2, only 10-9 M GnRH increased 3H-Gln LH and 35S-Met LH production, whereas in cells exposed to E2) all concentrations of GnRH (10-11-10-9 M) increased 3H-Gln LH and 35S-Met LH production. In all experiments, the specific activity of radiolabeled LH released under basal conditions was greatly reduced by stimulation with GnRH. These results suggest that GnRH regulates both LH glycosylation and LH polypeptide synthesis and that E2 lowers the physiological concentration of GnRH necessary to stimulate this biosynthetic response. Moreover, estrogenâ€™s enhancement of GnRH-stimulated LH release appears to be due to its action on mechanisms regulating the release of previously synthesized stored hormone as well as the release of newly synthesized LH.