To understand the mechanisms underlying mutagenesis in eukaryotes better, we have cloned mouse and human homologs of the Escherichia coli dinB gene. E. coli dinB encodes DNA polymerase IV and greatly increases spontaneous mutations when overexpressed. The mouse and human DinB1 amino acid sequences share significant identity with E. coli DinB, including distinct motifs implicated in catalysis, suggesting conservation of the polymerase function. These proteins are members of a large superfamily of DNA damage-bypass replication proteins, including the E. coli proteins UmuC and DinB and the Saccharomyces cerevisiae proteins Rev1 and Rad30. In a phylogenetic tree, the mouse and human DinB1 proteins specifically group with E. coli DinB, suggesting a mitochondrial origin for these genes. The human DINB1 gene is localized to chromosome 5q13 and is widely expressed.