Enzymatic photoreactivation is a DNA repair mechanism that removes UV-induced pyrimidine dimer lesions by action of a single enzyme, photolyase, and visible light. Its presence has been demonstrated in a wide variety of organisms, ranging from simple prokaryotes to higher eukaryotes. We have isolated a human gene encoding a 66-kDa protein that shows clear overall homology to known bacterial photolyase genes. The human gene product is more similar to plant blue-light receptors within class I photolyases than to higher eukaryote class II photolyases. Northern blot analysis showed two transcripts with constitutive expression in all tissues examined and an elevated expression in testis. In situ hybridization with a cDNA-derived probe localized this human gene to chromosome 12q23-q24.1. Southern analysis of the cloned human gene suggests a wide distribution of the gene family in various species.