We describe a method for isolating cosmid clones randomly from mouse chromosome 17. A cosmid library was constructed from the mouse-Chinese hamster cell line R4 4-1 that contains a limited amount of mouse DNA (chromosomes 17 and 18 and some other unidentified material) on a Chinese hamster background. The library was screened with the murine repetitive sequence probe pMBA14, which selectively hybridizes with mouse DNA. The mouse-derived cosmid clones thus identified were individually hybridized with DNA from the mouse-Syrian hamster cell line JS17 containing all mouse chromosomes except chromosome 17 on a Syrian hamster background. We deduced that the cosmid clones that contained sequences absent in JS17 were derived from mouse chromosome 17. One of the chromosome 17-derived cosmid clones, 3-4-1 (located proximal to the T122/T66C segment) was found to be highly polymorphic among European wild-mouse populations and may be a useful probe to elucidate the evolution and migration of Mus species. The randomly isolated mouse-derived cosmid clones can also be screened for the presence of functional genes. Using testicular cDNA as a probe, a testis-specific gene was cloned from mouse chromosome 17.