The DNA polymerase-alpha of Plasmodium falciparum was characterized according to aphidicolin sensitivity and immunological reactivity with monoclonal anti-sera against human DNA polymerase-alpha. Two major (105 and 72 kDa) and two minor (180 and 130 kDa) catalytic subunits of P. falciparum DNA polymerase-alpha were detected on activity gels. Activity gels did not indicate the presence of a DNA polymerase-beta in P. falciparum. Metabolically labeled polypeptides at 180, 105, 72, and 52 kDa were immunoprecipitated from Plasmodium nuclear extracts with the anti-KB cell DNA polymerase-alpha monoclonal antibody and, by size, correspond to the major subunits of mammalian DNA polymerase-alpha. The monoclonal antibody also neutralized Plasmodium DNA polymerase activity. Plasmodium DNA polymerase was synthesized predominantly at an early schizont stage at which time the parasite began to synthesize its DNA and multiply. No evidence for phosphorylation of the major catalytic subunit was obtained. Plasmodium growth, DNA synthesis, and DNA polymerase activity were inhibited significantly in parallel by aphidicolin. These results suggest that P. falciparum has a typical eukaryotic DNA polymerase-alpha and that regulation of its activity appears to be at the transcriptional level.