The von Hippel-Lindau (VHL) disease gene is a tumor suppressor located at 3p25-26. While amplifying intron 1 of this gene, a smaller-than-expected product was found. This fragment was sequenced and was approximately 78% similar in sequence to the VHL gene and completely lacked sequence from the intron. No stop codons were found in the sequenced region. Using this DNA fragment as a probe for Northern blot hybridization analysis, no evidence was found for expression of a unique RNA. Because of the lack of intron 1 sequence and the likely lack of expression, the new sequence is most probably a part of a VHL processed pseudogene. The putative pseudogene was mapped to human chromosome band 1q12 using the polymerase chain reaction with template DNA from human/rodent somatic cell hybrids, a radiation hybrid panel, and a set of primers that were chosen to be maximally divergent from the genuine VHL gene. The human/rodent somatic cell hybrid DNAs were then used on Southern blots to determine which human bands are from the pseudogene and which are from the functional gene. This knowledge is valuable in interpreting Southern blot evidence of VHL gene abnormalities.