We constructed a murine genomic DNA library in lambda EMBL3 and have isolated and determined the nucleotide sequence of the murine thyrotropin beta-subunit (TSH beta) gene. The cloned gene was derived from a thyrotropic tumor and had no detectable rearrangements when compared to the murine TSH beta gene in total genomic DNA. The murine TSH beta gene is 5 kb in size and consists of five exons and four introns. The 5' untranslated region of the mRNA is encoded except for a single nucleotide by exons 1, 2, and 3. The protein-coding regions are encoded by exons 4 and 5 while the 3' untranslated region is entirely contained in exon 5. Primer extension analysis using an exon 1-specific primer was used to map the 5' end of the gene. Two transcriptional start sites are present in the murine TSH beta gene which appear to be positioned by two TATAAA sequences located 40 bp apart. In all, 99% of transcripts initiate at the downstream site. Transcription from both start sites is affected by thyroidal status in both murine pituitaries and in TtT97 thyrotropic tumors. Finally, sequences homologous with putative thyroid-responsive elements and cyclic AMP-responsive elements are present in the 5'-flanking region and may be important in regulating negative and positive effects on TSH beta gene expression.