Based on deletion and complementation mapping and DNA sequencing, a new recessive fully penetrant mutation (DNApol-ϵpl10R), causing prolonged larval life and larval/early pupal lethality, is identified as the first mutant allele of the DNApol-ϵ (CG6768) gene of Drosophila melanogaster. A same-sense base pair substitution in exon 1 of the DNApol-ϵ gene is associated with retention of the first intron and significant reduction in DNApol-ϵ transcripts in DNApol-ϵpl10R homozygotes. Homozygous mutant larvae show small imaginal discs with fewer cells and reduced polyteny in salivary glands, presumably because of the compromised DNA polymerase function following exhaustion of the maternal contribution. Extremely small and rare DNApol-ϵpl10R homozygous somatic clones in DNApol-ϵpl10R/+imaginal discs confirm their poor mitotic activity. The DNApol-ϵpl10R homozygotes, like those expressing DNApol-ϵ-RNAi transgene, show high sensitivity to DNA damaging agents. The first mutant allele of the DNApol-ϵ gene will facilitate functional characterization of this enzyme in the genetically tractable Drosophila model.