DNA samples from about 100 human-hamster somatic cell hybrids, previously characterized by conventional banding techniques, were amplified with dual-Alu PCR. The products were then used as probes in FISH experiments on normal human metaphases for an accurate cytogenetic characterization of the human material retained in each hybrid. In addition to entire chromosomes, most hybrids were found to contain one or a few chromosome fragments, as a result of rearrangements that had occurred in vitro. Forty additional primary hybrids, in which conventional cytogenetic analysis failed to reveal any complete human chromosome, contained many human chromosome fragments. More than 300 chromosome fragments were scored and their precise chromosomal location recorded. We show data indicating that subchromosomal painting libraries generated from these hybrids can be favorably used in the fine characterization of chromosomal rearrangements encountered in clinical cytogenetics or in tumor cytogenetics, and in tracking chromosomal changes that occurred in primate evolution.