The murine gene encoding dihydrofolate reductase (DHFR) has been localized to a particular mouse chromosome by complementation mapping. Microcells prepared from diploid mouse fibroblasts were fused with mutant hamster cells lacking the dihydrofolate reductase gene (Dhfr), and DHFR+ microcell hybrids were selected in medium lacking purines and pyrimidines. The complemented hybrids expressed wild-type levels of DHFR enzyme activity, and selectively retained a single mouse chromosome--chromosome 13. Genomic Southern blots of DNAs prepared from these hybrids and from an independently derived collection of clones isolated without selection on the DHFR phenotype confirmed the assignment of murine Dhfr gene sequences to chromosome 13. This assignment provides further evidence for the existence of genetic homology between regions of mouse chromosome 13 and human chromosome 5. In support of this view, we show that the gene encoding hexosamindase B, a chromosome 5 marker in man, also maps to mouse chromosome 13.