A series of mouse-hamster somatic cell hybrids, containing reduced numbers of mouse chromosomes and a complete set of hamster chromosomes, was used to determine the chromosomal locations of the genes for the alpha and beta subunits of mouse thyrotropin. Cloned cDNA probes for each subunit, in conjunction with Southern blot analysis of DNA treated with the restriction enzyme BamHI, allowed for assignment of the alpha-subunit gene to mouse chromosome 4 and of the beta-subunit gene to chromosome 3. Mouse alpha-subunit gene sequences always segregated with chromosome 4 (concordant in 14 hybrids) and the enzyme markers phosphoglucomutase 2 and 6-phosphogluconate dehydrogenase. Mouse beta-subunit gene sequences always segregated with chromosome 3 (concordant in 15 hybrids). Thus, the genes for at least one of the glycoprotein hormones, thyrotropin, are on different chromosomes.