The mRNA and protein products produced by an alpha-subunit-secreting pituitary tumor were studied to further characterize the early steps in glycoprotein hormone biosynthesis. alpha-Subunit mRNA was readily detected by RNA blot hybridization to alpha-subunit cDNA and was qualitatively normal, with a length of about 800 bases. Although only excess free alpha-subunit was secreted by the tumor, abundant beta-subunit mRNA hybridized to a cDNA which recognized both LH beta and CG beta sequences. Surprisingly, the beta-subunit mRNA in the pituitary tumor was characteristic of CG beta mRNA. The beta-subunit mRNA migrated in parallel with placental CG beta mRNA (approximately 1000 bases) and was easily resolved from LH beta mRNA (approximately 700 bases). Using site-specific hybridization and oligonucleotide-primed extension, the beta-subunit mRNA in pituitary tumor was found to have an extended 5'-untranslated tract (366 bases) characteristic of CG beta gene transcripts rather than LH beta gene transcripts, in which the 5'-untranslated tract is only 9 bases. Activation of the CG beta-like promoter rather than the LH beta promoter indicates that the cellular mechanisms regulating tissue-specific expression of the beta-subunit gene were altered by the neoplastic changes in the pituitary tumor. Protein analysis of tumor homogenate demonstrated large amounts of alpha-subunit in the form of intact FSH and small amounts of intact LH and free alpha-subunit. Despite the presence of abundant CG beta-like mRNA in the tumor, intact hCG or hCG beta was not detected. The uncoupling of beta-subunit gene expression and protein biosynthesis in the setting of ongoing alpha-subunit biosynthesis provides a potential mechanism for unbalanced synthesis and secretion of free alpha-subunit.