Two distinct DNA polymerases (DNA nucleotidyltransferases) have been partially purified from exponentially growing cells of the slime mold Dictyostelium discoideum. The major DNA polymerase activity, DNA polymerase A, has physical and enzymatic properties similar to DNA polymerase alpha from vertebrate cells. DNA polymerase B, representing less than 10% of the polymerase activity in homogenates, is similar to DNA polymerase beta from vertebrate cells in that its activity is not inhibited by N-ethylmaleimide, aphidicolin, or KCl at a concentration below 200 mM. Like DNA polymerase beta, the activity of Dictyostelium DNA polymerase B is sensitive to 2', 3'-dideoxythymidine 5'-triphosphate. However, it differs from vertebrate DNA polymerase beta in that it chromatographs as an acidic protein and has an apparent molecular weight of 70,000. Previous reports of the absence of a beta-like DNA polymerase in D. discoideum apparently resulted from inadequate extraction and assay conditions for the enzyme. It seems quite probable from the results of this study and those of other studies (e.g., yeast and Tetrahymena pyriformis) that lower eukaryotic organisms, as well as vertebrates and prokaryotes, contain more than one form of DNA polymerase.