The glycoprotein hormones consist of a common alpha-subunit and distinct, but structurally related, beta-subunits which confer biological specificity. To study glycoprotein hormone gene expression, we prepared specific oligonucleotides complementary to nonhomologous regions of the alpha-subunit and each of the beta-subunit mRNAs encoding human LH, CG, TSH, and FSH. beta-Subunit mRNAs were expressed at relatively low levels in normal pituitary tissue, but were found in greater amounts in pituitary gonadotroph and thyrotroph adenomas. The lengths of the glycoprotein hormone alpha- and beta-subunit mRNAs in normal and neoplastic pituitary tissue were indistinguishable. Expression of different members of the closely related LH beta/CG beta gene family were examined in normal and neoplastic pituitary and placenta using short oligonucleotides complementary to nonhomologous regions of the genes. Although CG beta mRNA was found previously in a pituitary adenoma, none was detected in normal pituitary tissue. In placenta, there was abundant expression of the CG beta gene, but no expression of the LH beta gene, consistent with the acquisition of tissue-specific regulatory sequences in the recently evolved upstream promoter recognition site of the CG beta gene. Primer extension analysis of CG beta mRNA indicated that the same CG beta gene promoter site was used in both normal placenta and JEG-3 choriocarcinoma cells. Of the two CG beta genes that have been reported to be functional, CG beta gene 5 was preferentially expressed in both normal placenta and the neoplastic JEG-3 cell line. CG beta gene 3 expression accounted for only about 5% of the total CG beta mRNA. The previously uncharacterized human TSH beta and FSH beta mRNAs were studied in normal and neoplastic pituitary tissue. At least two species of FSH beta mRNA were found on Northern blots. Oligonucleotide-primed extension of pituitary mRNA demonstrated that FSH beta mRNA heterogeneity resulted from transcription from distinct promoter sites, encoding 5'-untranslated tracts of 48 and 83 bases. These studies demonstrate that specific oligonucleotide probes distinguish expression of the structurally related glycoprotein hormone beta-subunit mRNAs, allowing analyses of tissue-specific gene expression under different physiological conditions as well as in normal and neoplastic tissues.