Long-term effects of gonadotropin-releasing hormone (GnRH) and/or estrogen on pituitary mRNA levels for the β-subunit of luteinizing hormone (LH-β) were determined in anterior pituitary glands from ovariectomized (OVX) ewes. The relative roles of these two factors were assessed by studying hypothalamopituitary disconnected (HPD) ewes with appropriate hormonal treatments. Levels of LH-β mRNA were increased by ovariectomy and substantially reduced by HPD. Treatment of OVX-HPD ewes with pulses of GnRH (250 ng each 2 h) for 1 week restored LH-β mRNA levels to OVX levels, whereas treatment with estrogen alone did not alter the low levels found in OVX-HPD ewes. Combined GnRH and estrogen treatment for one week produced LH-β mRNA levels that were similar to those found in OVX-HPD ewes given GnRH alone; plasma LH pulse amplitudes were also similar in these two groups. From these data we conclude that the long-term negative feedback effect of estrogen to reduce LH secretion is due to a primary inhibition of GnRH secretion and is not a pituitary effect of estrogen. Long-term regulation of LH-β mRNA is thus primarily regulated by GnRH.