Previous studies have shown that insulin augments GnRH-stimulated LH synthesis and release from primary gonadotrophs. In this study, regulation of LHbeta gene expression by GnRH and insulin was examined in LbetaT2 cells. Endogenous LHbeta mRNA is stimulated 2.4-fold by insulin alone, 2.6-fold by GnRH alone, and 4.7-fold by insulin together with GnRH. This effect of insulin, like GnRH, mapped to sequences -140 to +1 in the mouse LHbeta gene. Insulin together with GnRH stimulates activity of an LHbeta-reporter gene 7.1-fold; whereas, GnRH alone or insulin alone stimulates the reporter activity 2.8- and 3.1-fold, respectively. Blocking the binding of Egr-1 to sequences -51 to -42 in the LHbeta gene inhibits effects of insulin and GnRH. Insulin together with GnRH increases Egr-1 mRNA levels and total Egr-1 binding to LHbeta DNA. These findings indicate that insulin may impact regulation of the reproductive axis at the level of the pituitary.