To evaluate the hypothalamic contribution to the development of anterior pituitary (AP) cells we surgically disconnected the hypothalamus from the pituitary (hypothalamo-pituitary disconnection, HPD) in fetal sheep and collected pituitaries 31 days later. Pituitaries (n = 6 per group) were obtained from fetal sheep (term = 147 +/- 3 days) at 110 days (unoperated group) of gestation and at 141 days from animals that had undergone HPD or sham surgery at 110 days. Cells were identified by labelling pituitary sections with antisera against the six AP hormones. Additionally, we investigated the colocalization of glycoprotein hormones. The proportions of somatotrophs and corticotrophs were unchanged by age or HPD. Lactotrophs increased 80% over time, but the proportion was unaffected by HPD. Thyrotrophs, which were unaffected by age, increased 70% following HPD. Gonadotrophs increased with gestational age (LH+ cells 55%; FSH+ cells 19-fold), but this was severely attenuated by HPD. We investigated the possible existence of a reciprocal effect of HPD on multipotential glycoprotein-expressing cells. Co-expression of LH and TSH was extremely rare (< 1%) and unchanged over the last month of gestation or HPD. The increase of gonadotrophs expressing FSH only or LH and FSH was attenuated by HPD. Therefore, the proportions of somatotrophs, lactotrophs and corticotrophs are regulated independently of hypothalamic input in the late gestation fetal pituitary. In marked contrast, the determination of the thyrotroph and gonadotroph lineages over the same time period is subject to complex mechanisms involving hypothalamic factors, which inhibit differentiation and/or proliferation of thyrotrophs, but stimulate gonadotrophs down the FSH lineage. Development of a distinct population of gonadotrophs, expressing only LH, appears to be subject to alternative mechanisms.