alpha-Satellite subfamilies from chromosomes 21 and 13 are almost identical in sequence and cannot be distinguished from each other by hybridization techniques. A general method based on membrane-bound PCR is described here, allowing the discrimination of alpha-satellite DNA sequences from each of these two chromosomes, after detection by Southern blot hybridization. The PCR conditions were developed using somatic hybrid DNAs. The method was tested in membrane-bound PCR by using the alpha-satellite bands from a Southern blot of a CEPH family. The chromosomal origin of these bands, previously determined by linkage analysis, was confirmed by this method.