The current studies reveal that expression of the mouse TSH beta-subunit gene in the pituitary gland and TtT97 murine thyrotropic tumor results in multiple mRNAs which arise by alternative splicing. This was deduced by 1) sequencing the extended products of TSH beta mRNA primed with an oligonucleotide complementary to the protein coding region and 2) demonstrating primer extension of oligonucleotides complementary to the optional exon junctions. These results, when correlated with the sequence of the TSH beta gene, revealed a structure comprising five exons and four introns. Exon 1, which is common to all of the mRNAs, and optional exons 2 and 3 code only for 5'-untranslated (UT) sequences, whereas exons 4 (includes one nucleotide of 5'-UT) and 5 encode the protein coding and 3'-UT regions. Multiple splicing events generate mRNAs with 5'-UT regions of 28, 69, 75, and 116 nucleotides which include exon 1, exons 1 and 3, exons 1 and 2, and exons 1, 2, and 3, respectively. In contrast, in the hypothyroid rat pituitary messages possessing optional exons were not detected, and thus the TSH beta mRNA with a 5'-UT region of 28 nucleotides predominated. We identified an additional transcriptional start site located 40 nucleotides upstream in both the mouse and rat TSH beta gene. Optional exon splicing was not detectable in transcripts initiated from the upstream start site of the murine gene. In the hypothyroid state, transcripts initiated at the upstream site were considerably less abundant than those initiated at the downstream site. Transcription from both start sites was inhibited by thyroid hormone in both mice and rats.