Whether estradiol targets a subpopulation of gonadotrope cells was investigated in this study. Ovariectomized ewes (OVX) or OVX ewes immunized against GnRH and treated with hourly pulses of GnRH analogue (OVX-IMG) were killed at 6, 12, 16, and 24 h after administration of 50 μg of 17β-estradiol (E₂). Control ewes received no E₂ treatment. In OVX or OVX-IMG ewes killed 6 h after E₂ injection, a decrease in gonadotropin plasma levels was observed compared with non-E₂-treated ewes. In contrast, a surge in gonadotropin plasma concentrations occurred in ewes killed 16 h after injection. The percentage of total immunoreactive gonadotrope cells among the pituitary cells was lower in E₂-treated ewes compared with nontreated animals. The proportion of monohormonal LH cells was constant throughout the experiment, except at the surge peak, where it was enhanced. In the OVX ewes, the proportion of bihormonal LH/FSH cells was lower in the E₂-treated ewes compared to the nontreated ewes (P < 0.001), with a more pronounced decrease 16 h after E₂ injection. A slight increase occurred 12 h after E₂ injection compared with 6 h after injection (P < 0.05). A similar pattern was observed in the OVX-IMG ewes, except at 12 h after E₂ injection, when no increase occurred. In both OVX and OVX-IMG ewes, injection of E₂ decreased FSHβ mRNA expression but did not alter the relative levels of LHβ mRNA. These data suggest that the negative feedback of E₂ on LH and FSH secretion mainly targets the bihormonal cells and occurs, at least in part, directly at the pituitary level. During the gonadotropin surge, the sustained FSH release from the bihormonal cells would induce a switch from bihormonal cells to monohormonal LH cells by depleting these cells of FSH.