Comparative mapping of human and mouse DNA for regions of genetic homology between human Chromosome 21 and the mouse genome is of interest because of the possibility of developing mouse models of human trisomy 21 (Down syndrome), understanding chromosome evolution, and isolating novel sequences conserved between the two species. At least two mouse chromosomes are known to carry sequences homologous to those on human Chromosome 21: mouse Chromosome 16 (D21S16h, D21S13h, D21S52h, App, Sod-1, Mx-1, Ets-2, Prgs,Ifnar) and mouse Chromosome 17 (D21S56h, Crya-1, and Cbs). Recently, five additional genes have been mapped within region 21q22 of human Chromosome 21:PFKL, CD18, COL6A1, COL6A2, and S100B. To assign these sequences to specific mouse chromosomes, we used human cDNA probes for COL6A1, COL6A2, CD18, and PFKL and a rat brain cDNA probe for S100B in conjunction with a panel of seven Chinese hamster-mouse somatic cell hybrids segregating mouse chromosomes. The specific chromosome complements of the hybrid cell lines and the presence or absence of hybridizing mouse sequences in their DNAs allow us to assign all five sequences to mouse Chromosome 10, with the assignment of Pfkl reported here for the first time. Analysis of genomic mouse DNA fragments produced by digestion with rare-cutting restriction enzymes and separated using pulsed-field gel electrophoresis allows us to construct a fine-structure physical map of two segments of the region of Chromosome 10 containing these five markers. The five loci span at least 1900 kb of mouse DNA and are consistent with the human order: Pfkl-Cd-18-Col6a-1-Col6a-2-S100b.(ABSTRACT TRUNCATED AT 250 WORDS)