DNA primase activlty has been resolved from a purified DNA primase-polymerase α complex of HeLa cells by hydrophobia affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase α. The free DMA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyaorylanide gel electrophoretio analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velcity sedimentation has an S20, W of 5. DNA primase synthesizes RNA ollgomers with single- stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase α in a manner that has already been described for several purified eukaryotic DNA primase-polymerase α complexes. The purified free DNA prinase activity is resistant to neutralizing anti-human DNA polymerase α antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase α and also DNA polymerase α complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase α. Taken together these results indicate that the DNA primase and polymerase α activities of the DNA primase-polymerase α complex reside on separate polypeptides that associate tightly through hydrophobio Interactions.