The gene for the beta-subunit of follicle-stimulating hormone (FSH-beta) was isolated from a library of bovine DNA fragments cloned in bacteriophage gamma and the complete nucleotide sequence of the gene was determined. The bovine FSH-beta gene contains approximately 4000 nucleotides and consists of three exons separated by two intervening sequences. The transcription initiation site of the gene was mapped by nuclease protection experiments. Analysis of RNA species present in pituitary mRNA demonstrated the presence of a 4.0-kb RNA containing FSH-beta sequences, which is the appropriate size for the primary transcript of the gene. Comparison of nucleotide sequence of the 5'-flanking sequence of the FSH-beta gene to the 5'-flanking regions of other pituitary glycoprotein hormone genes reveals little sequence similarity.