We previously isolated two polymorphic chromosome 21q probes, pVC1.21c (D21S190) and pVC1.34a (D21S149), localized in 21qcen-21q21.2. In addition, pVC1.21c recognized a sequence in 21q22.1-q22.2 and both probes cross-hybridized with non-chromosome-21 sequences. In this study we refined the proximal 21q locations of probes pVC1.21c and pVC1.34a to 21q11.1 and demonstrated that they recognize sequences on chromosome 13 but not on chromosomes 14, 15, and 22. Furthermore, the polymorphisms associated with the two loci were assigned to pericentromeric 13q for pVC1.34a and distal 21q for pVC1.21c. Our results are indicative of a region of unique sequence homology in the pericentromeric region of the long arms of chromosomes 13 and 21.