The preparation and properties of an antiserum to human DNA polymerase I (6 to 8 S) are described. Care was taken in the purification of the antigen to remove certain other DNA polymerases found in human cells. An incubation of antigen and antiserum lasting about 48 hours is necessary to achieve maximal inhibition. About 1 mug of the antipolymerase immunoglobulin G, prepared in rats, neutralizes 60% of the activity present in 54 ng of the enzyme. Tritrations varying both antiserum and enzyme demonstrate clear regions of antigen and antibody excess. Inhibition of enzyme activity is about the same whether the templateprimer is (dA)n-(dT)12-18, or partially digested DNA. An assay was developed which measures the remaining activity in the supernatant after precipitation of enzyme-antibody complexes with goat anti-rat immunoglobulin G. In this assay, 2.2 mug of the antipolymerase immunoglobulin G quantitatively bind 33 ng of DNA polymerase I. With use of the direct neutralization assay and the immuno-precipitation test, we found little, if any, antigenic relationship between DNA polymerase I and DNA polymerase II (3.4 S). Similarly, little, if any, relationship was found to the DNA polymerases from five RNA tumor viruses. The activities of RNA-directed DNA polymerases from the blood leukocytes of two patients with acute myelogenous leukemia and from the placentas of rhesus monkeys were not inhibited in neutralization assays which were shortened because these enzymes were thermolabile. In identically shortened neutralization assays, the antipolymerase immunoglobulin G neutralized up to 76% of the activity of DNA polymerase I. In addition to its utility in distinguishing cellular DNA polymerases, the rat antiserum should be useful reagent for testing of novel DNA polymerases isolated in small quantities from human tumors for contamination with DNA polymerase I. This enzyme is present in abundance in proliferating tissue and often confuses the biochemical characterization of these novel enzymes.