The mitogenic and regulatory effects of estrogen (E₂) in adenohypophysial cells are known to be mediated through the nuclear estrogen receptor (ERα). Expression of ERα and several of its messenger ribonucleic acid (RNA) alternate splice variants has been shown to be restricted to prolactinomas and gonadotroph tumors. However, little is known about gene expression patterns of the novel nuclear hormone receptor ERβ in the neoplastic pituitary. ERβ has high homology to ERα in the DNA- and ligand-binding domains, but encodes a distinct transcriptional activating function-1 (AF-1) domain. Using RT-PCR analysis of total RNA from 38 human pituitary adenomas, we found that ERβ messenger RNA was coexpressed with ERα and its splice variants in 60% of prolactinomas, 100% of mixed GH/PRL tumors, and 29% of gonadotroph tumors. ERβ gene expression was not limited to ERα-positive tumor subtypes, however, and was also found in 100% of null cell tumors, 80% of somatotroph tumors, and 60% of corticotroph tumors. Because ERβ is coexpressed with ERα and its splice variants in prolactinomas and gonadotroph tumors, we functionally characterized the potential interactions between ERβ and ERα. We also examined the potential cooperative effects on ERβ-mediated gene expression of a tumor-specific truncated Δ5ERα splice variant that has been shown to be coexpressed in the majority of ERα-positive tumors. This exon 5 splice variant encodes the AF-1 domain as well as regions critical for DNA binding and nuclear localization, but lacks the ligand-binding and AF-2 domains. Mammalian expression vectors encoding ERα, Δ5ERα, and/or ERβ complementary DNAs were transiently transfected along with an E₂ response element promoter-luciferase (ERELuc) reporter into human ERα/ERβ-negative osteosarcoma U2-OS cells. ERβ was less potent than ERα in activating E₂-stimulated ERELuc activity (4- vs. 14-fold relative to basal control levels). However, when Δ5ERα was coexpressed with ERβ or ERα, E₂-stimulated ERELuc activity was markedly increased to 8- and 57-fold, respectively, relative to basal control levels when each full-length isoform was expressed alone. Finally, coexpression of ERβ with ERα did not significantly alter the E₂-stimulated ERELuc activity induced by ERα alone. Cotreatment with tamoxifen markedly inhibited all E₂-stimulated ERELuc responses to baseline levels. Together, these data suggest that ERβ has a minor role in mediating E₂ responses in ERα-positive tumors, but may be the main mediator of E₂-stimulated gene expression when expressed alone in somatotroph, corticotroph, and null cell tumors. This low, but significant, level of ERβ trans-activation potential may be enhanced by coexpression of Δ5ERα in neoplastic pituitary. Therefore, E₂-mediated gene expression in normal and neoplastic pituitary appears to be highly dependent on the expression of ERα and ERβ isoforms, which have varying transcriptional activities.