The mosquito homolog of mammalian DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen (PCNA)-independent form of DNA polymerase delta, has been purified from mosquito larval extracts. The polymerase epsilon was separated from DNA polymerase alpha by chromatography on hydroxylapatite, and the enzyme was subsequently purified on single-stranded DNA agarose, followed by a 5' AMP-agarose chromatography step. The purified polymerase exhibits an intrinsic 3'-5' exonuclease activity and shows high activity using an oligo-primed DNA template. Neither human nor Drosophila PCNA stimulated this polymerase activity. Additional immunochemical and biochemical evidence indicates that this enzyme is distinct from DNA polymerase alpha.