TheSaccharomyces cerevisiae RAD30gene encodes DNA polymerase η. Humans possess two Rad30 homologs. One (RAD30A/POLH) has previously been characterized and shown to be defective in humans with the Xeroderma pigmentosum variant phenotype. Here, we report experiments demonstrating that the second human homolog (RAD30B), also encodes a novel DNA polymerase that we designate polι. polι, is a distributive enzyme that is highly error-prone when replicating undamaged DNA. At template G or C, the average error frequency was ∼1 × 10⁻². Our studies revealed, however, a striking asymmetry in misincorporation frequency at template A and T. For example, template A was replicated with the greatest accuracy, with misincorporation of G, A, or C occurring with a frequency of ∼1 × 10⁻⁴to 2 × 10⁻⁴. In dramatic contrast, most errors occurred at template T, where the misincorporation of G was, in fact, favored ∼3:1 over the correct nucleotide, A, and misincorporation of T occurred at a frequency of ∼6.7 × 10⁻¹. These findings demonstrate that polι is one of the most error-prone eukaryotic polymerases reported to date and exhibits an unusual misincorporation spectrum in vitro.