Estrogen receptor-(ER) levels in gonadotropes are increased during the follicular phase of the ovine estrous cycle, a time of increased frequency of pulsatile secretion of gonadotropin releasing hormone and elevated plasma estrogen levels. In the present study, our first aim was to determine which of these factors causes the rise in the number of gonadotropes with ER-. Ovariectomised (OVX) hypothalamo-pituitary disconnected (HPD) ewes (n = 4-6), received the following treatments: 1. No treatment; 2. Injection (i.m.) of 50 µg estradiol benzoate (EB); 3. Pulses (300ng i.v.) of GnRH every 3 h; 4. GnRH treatment as in group 3 and EB treatment as in group 2; 5. Increased frequency of GnRH pulses commencing 20 h before termination; 6. GnRH treatment as in group 5 with EB treatment. These treatments had predictable effects on plasma LH levels. The number of gonadotropes in which ER was present (by immunohistochemistry) was increased by either GnRH treatment or EB injection, but combined treatment had the greatest effect. Immunohistochemistry was also performed to detect phosphorylated CREB (pCREB) and Fos protein in gonadotropes. The number of gonadotropes with Fos and with pCREB was increased only in Group 6. We conclude that either estrogen or GnRH can up-regulate ER- in pituitary gonadotropes. On the other hand, during the period of positive feedback action of estrogen, the appearance of pCREB and Fos in gonadotropes requires the combined action of estrogen and increased frequency of GnRH input. This suggests convergence of signaling for GnRH and estrogen.