Somatic cell genetic techniques were used to establish linkage assignments for the loci coding for galactokinase (GLK) and thymidine kinase (TK) in the laboratory mouse, Mus musculus.Four fusion experiments produced cell hybrids that segregated mouse chromosomes. Three series of hybrid clones were produced from the fusion of mouse macrophages or fibroblasts and Chinese hamster cells of the E36 line, which is deficient in hypoxanthine phosphoribosyltransferase activity. Clones were assayed for the expression of 11 mouse isozymes coded by genes on 11 chromosomes. Ten isozymes were retained at high frequencies, but the mouse GLK phenotype was absent in most primary clones and all secondary clones. Chromosome analysis of 24 of these hybrids indicated that GLK activity was lost concordantly with chromosome 11. One hybrid clone was produced from the fusion of peritoneal macrophages and hamster cells of the a3 line, which lacks TK activity. Mouse GLK activity was expressed in this primary clone and all secondary clones isolated in HAT medium, and was lost in all secondary clones backselected in medium with 5-bromodeoxyuridine. Thus, the genes coding for GLK and TK are syntenic and both can be assigned to chromosome 11. The absence of chromosome 11 in the related series of a3 hybrids, and its rapid segregation in 3 series of E36 hybrids, suggests that it carries a third locus (or loci), the retention of which is detrimental to Chinese hamster/mouse hybrids.