Two DNA polymerase activities, polymerases A and B, were separated from the Triton-treated cell homogenate of exponentially growing Tetrahymena pyriformis by phosphocellulose column chromatography. Their properties were as follows. Polymerase A: The molecular weight was about 140,000, the sedimentation value was about 6.2S, the optimum Mg2+ concentration was 15 mM, the optimum K+ (or Na+) concentration was 20 mM, and the optimum pH was 7.4. The enzyme activity was inhibited by cytosine-beta-D-arabinofuranoside-5'-triphosphate (araCTP) or aphidicolin, but not by 2'-3'-dideoxythymidine-5'-triphosphate (ddTTP). Polymerase B: The molecular weight was about 70,000, the sedimentation value was 4.3S, the optimum Mg2+ concentration was 15 mM, the optimum K+ (or Na+) concentration was 150 mM, and the optimum pH was 8.4. The enzyme activity was inhibited by ddTTP, but not by araCTP or aphidicolin. Polymerases A and B were both found to be N-ethylmaleimide-sensitive. These results indicate that at least two N-ethylmaleimide-sensitive DNA polymerases, A and B, are present in exponentially growing Tetrahymena cells. Polymerase A bears many similarities to DNA polymerase alpha of higher eukaryotes and polymerase B also bears similarities to DNA polymerase beta except as regards N-ethylmaleimide sensitivity. Based on the properties of polymerases A and B, the relation of Tetrahymena DNA polymerases reported by several investigators is discussed.