Base excision repair is one of the major mechanisms by which cells correct damaged DNA. We have developed an in vitro assay for base excision repair which is dependent on a uracil-containing DNA template. In this report, we demonstrate the fractionation of a human cell extract into two required components. One fraction was extensively purified and by several criteria shown to be identical to DNA polymerase beta (Polbeta). Purified, recombinant Polbeta efficiently substituted for this fraction. Escherichia coli PolI, mammalian Poldelta and to a lesser extent Polalpha and epsilon also functioned in this assay. We provide evidence that multiple polymerases function in base excision repair in human cell extracts. A neutralizing antibody to Polbeta, which inhibited repair synthesis catalyzed by pure Polbeta by approximately 90%, only suppressed repair in crude extracts by a maximum of approximately 70%. An inhibitor of Polbeta, ddCTP, decreased base excision repair in crude extracts by approximately 50%, whereas the Polalpha/delta/epsilon inhibitor, aphidicolin, reduced the reaction by approximately 20%. A combination of these chemical inhibitors almost completely abolished repair synthesis. These data suggest that Polbeta is the major base excision repair polymerase in human cells, but that other polymerases also contribute to a significant extent.