A DNA polymerase with properties similar to mammalian polymerase beta has been isolated to near homogeneity from embryos of Drosophila melanogaster. A combination of exclusion chromatography and sodium dodecyl sulfate-gel electrophoresis indicates that this enzyme is composed of a single polypeptide of molecular weight-110,000. Optimum activity on a nicked template occurs at pH 8.4 in the presence of 15 mM MgCl2 and 250 mM NaCl. Enzyme activity is strongly inhibited by dideoxythymidine triphosphate but is relatively insensitive to aphidicolin and N-ethylmalemide. These properties clearly distinguish this enzyme from polymerase alpha, which has previously been characterized from this tissue. This report represents the first extensive purification of a beta-like polymerase from the Protostomic branch of the animal phylogenetic tree. It furthermore generates the potential for a genetic analysis of the function of polymerase beta in DNA recombination, repair, and synthesis.