Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that is comprised of two dissimilar subunits, alpha and beta, encoded by separate genes. We have isolated the gene encoding the beta-subunit of rat FSH by screening a rat genomic DNA-library by filter hybridization with bovine FSH-beta cDNA. Southern blot analysis of rat genomic DNA suggests that there is a single copy of the FSH-beta gene per haploid genome in the rat. The nucleotide sequence of the rat FSH-beta gene was determined and the amino acid sequence of the subunit was deduced. The gene is composed of three exons and two introns. The predicted amino acid sequence reveals that there is a 20-amino-acid signal peptide followed by a mature protein of 110 amino acid residues. Exons I, II, and III are 36, 187, and 1221 bp, respectively. Intron 1 (640 bp) interrupts the 5'-untranslated (UT) region (61 bp) and intron 2 (approximately 1 kb) interrupts the coding region between amino acid residues +34 and +35. Comparison of the amino acid sequence to those of the human and bovine FSH-beta subunits reveals 80% similarity to both species. The "CAGY" sequence, or the Cys-Ala-Gly-Tyr quartet of amino acids encoded by exon II is present in every other glycoprotein beta-subunit sequenced thus far, is altered in rat FSH-beta, with the Ala residue replaced by Glu. Primer extension analysis demonstrated that there is a single transcriptional start site. The nucleotide sequence of the 5'-flanking region (1 kb) was determined and compared to the nucleotide sequences of corresponding regions in the bovine and human genes. This analysis revealed that there are three regions in the 5'-flanking region of the rat FSH-beta gene that display greater than 80% sequence similarity to regions in the bovine and human genes. The second of these regions also shares similarity to segments within the 5'-flanking regions of the rat alpha- and LH beta-subunit genes. The characterization of the rat FSH-beta gene will enable further study of the regulation of rat FSH using in vitro systems.