The physiological effects of estrogen on the pituitary, including cellular proliferation and regulation of hormone synthesis, are mediated by the nuclear estrogen receptor (ER). The ER acts as a dimer to modulate gene transcription and contains specific functional domains encoded in different exons. Two separate, but related, forms of the receptor (ERalpha and ERbeta) exist, with distinct tissue and cell patterns of expression. Additional ER isoforms, generated by alternative messenger ribonucleic acid (mRNA) exon splicing, have been defined in several tissues and are postulated to play a role in tumorigenesis or in modulating the estrogen response. We examined 71 human pituitary adenomas of varying phenotypes and 6 normal pituitary specimens for ER mRNA forms by RT-PCR and hybridization blotting analysis. All prolactinomas (n = 14) contained ERalpha, and several contained ERbeta (5 of 14) mRNA. In comparison, 6 tumors that expressed PRL and GH expressed ERbeta (4 of 6) more frequently than ERalpha (3 of 6). ERbeta mRNA was also found more frequently in null cell (8 of 24 ERalpha and 14 of 24 ERbeta) and gonadotrope (13 of 21 ERalpha and 18 of 21 ERbeta) tumors. Additionally, ERbeta was found in 4 of 6 tumors that contained only GH, although ERalpha was not observed in this tumor type. Expression of the two ER forms within a tumor type was overlapping, but some tumors contained only 1 isoform. Expression of ERalpha mRNA splice variants also varied with cell type. All normal pituitaries contained ERalpha deletions of exon 4, 5, and 7, whereas only 2 of 6 samples contained the exon 2 deletion variant. Although the same ERalpha mRNA variants were observed among the various tumor types, the proportion of specific splice variants expressed varied. For example, most ER-positive prolactinomas expressed ERalpha variants with deletions of exon 2, 4, or 5, whereas gonadotropin tumors preferentially expressed the ERalpha exon 7 deletion variant. A novel ERbeta mRNA splice variant, missing exon 2, was observed in a majority of all ERbeta-positive tumors. Immunoblotting analysis of ERalpha and ERbeta proteins supported the mRNA results. Because ERalpha and ERbeta have different biological responses to selective ER modulators, and the ER deletion variants have biological effects distinct from those of the full-length ER, expression of these isoforms may influence the biological properties of these tumors and affect their ability to respond to estrogen and antiestrogen therapies.