Chromosome mapping by Southern analysis of DNA from somatic cell hybrids is often unsuccessful when only cDNA probes are available. p68 is a putative RNA helicase that is antigenically related to the simian virus 40 large tumor antigen. By using the polymerase chain reaction and oligode-oxynucleotide primers based on the cDNA sequence, we have identified introns in the p68 gene. Comparison of human and mouse DNA fragments amplified with these primers revealed length differences that allowed us to identify the human gene. Application of this technique to DNA from human-mouse somatic cell hybrids and cell lines derived from them by chromosome-mediated gene transfer allowed us to map p68 to the distal part of the long arm of chromosome 17.