Different subfragments of a cDNA coding for DNA topoisomerase I were used as probes to determine the chromosomal localization of topoisomerase I sequences in human cells. Southern blotting of restricted DNA from a panel of rodent-human somatic cell hybrids revealed the localization of the complete gene on chromosome 20 and the presence of two truncated topoisomerase I pseudogene sequences on chromosomes 1 and 22. In situ chromosome hybridization experiments confirmed these results showing the location of the complete gene on band q11.2-13.1 of chromosome 20, and the location of the pseudogene sequences on band q23-24 of chromosome 1 and q11.2-13.1 of chromosome 22.