A new gene (POLL), has been identified encoding the novel DNA polymerase lambda and mapped to mouse chromosome 19 and at human chromosome 10. DNA polymerase lambda contains all the critical residues involved in DNA binding, nucleotide binding, nucleotide selection, and catalysis of DNA polymerization and has been assigned to family X based on sequence homology with polymerase beta, lambda, mu, and terminal deoxynucleotidyltransferase. Here we describe a purification of DNA polymerase lambda from calf thymus that preferentially can replicate damaged DNA. By testing polymerase activity on non-damaged and damaged DNA, DNA polymerase lambda was purified trough five chromatographic steps to near homogeneity and identified as a 67-kDa polypeptide that cross-reacted with monoclonal antibodies against DNA polymerase beta and polyclonal antibodies against DNA polymerase lambda. DNA polymerase lambda had no detectable nuclease activities and, in contrast to DNA polymerase beta, was aphidicolin-sensitive. DNA polymerase lambda was a 6-fold more accurate enzyme in an M13mp2 forward mutation assay and 5-fold more accurate in an M13mp2T90 reversion system than human recombinant DNA polymerase beta. The biochemical properties of the calf thymus DNA polymerase lambda, described here for the first time, are discussed in relationship to the proposed role for this DNA polymerase in vivo.