The well documented mitogenic and hormone regulatory effects of estrogen (E2) on pituitary cells are mediated via its nuclear receptor (ER), a cellular homolog of v-erbA oncogene. ER isoforms generated by alternative exon splicing, termed ER variants delta 2ER to delta 7ER, have been identified in breast cancer and have been postulated to have important pathogenetic and clinical implications in tumorigenesis and/or development of hormone resistance. Because pituitary tumors, particularly prolactinomas, are known to be E2-dependent, we investigated alternatively spliced ER variant messenger ribonucleic acid expression in 40 human pituitary tumors of various phenotypes and normal pituitary tissues, using reverse transcription-PCR and Southern blot analyses. Nine of 11 prolactinomas readily expressed multiple ER variants (delta 2ER, delta 4ER, 5ER, and delta 7ER), whereas 6 of 11 tumors showed faint expression of delta 3ER. Four of 7 glycoprotein hormone-producing tumors that synthesized FSH beta expressed delta 2ER, delta 5ER, and delta 7ER. In 9 GH- and 10 ACTH-secreting tumors examined, the expression of normal and variant ER was restricted to tumors that also exhibited scattered PRL immunoreactivity. Variant and normal ER were not found in three null cell tumors (oncocytomas) that showed negative immunoreactivity for all pituitary hormones or their subunits. In contrast, only delta 4ER and delta 7ER were uniformly detected in normal pituitaries. delta 6ER was not detected in any normal or neoplastic pituitary specimen studied. We conclude that multiple alternatively spliced ER variants are coexpressed with normal ER in a tumor phenotype-specific manner. In addition, ER variants delta 2ER and delta 5ER were found to be tumor specific. Future functional studies will be required to determine whether coexpression of multiple ER variants along with normal ER confers a pathophysiological role in pituitary hormone regulation and/or tumor cell proliferation.