Human-rodent hybrid cell lines have been analyzed with regard to their human DNA content by using various DNA probe sets, derived from the hybrids, for in situ hybridization to normal human metaphase chromosome spreads. Total genomic hybrid DNA was compared with probe sets of hybrid DNA that were highly enriched in human sequences. The latter probes were obtained by amplification through the polymerase chain reaction (PCR) using oligonucleotide primers directed to human specific subsequences of the interspersed repetitive sequences Alu and L1. Previously unidentified chromosomal material within hybrid lines was characterized with speed and precision. It is demonstrated that the complete human complement of hybrid lines can be rapidly assessed by comparing the data obtained with the Alu-PCR products with the results from the L1-PCR products or from the genomic hybrid DNA. This approach using interspersed repetitive sequence-PCR products is simple and fast and also provides an alternative way of generating complex DNA probe sets for the specific delineation of entire chromosomes or subchromosomal regions by in situ hybridization.