Poly(ADP-ribose) polymerases from Ehrlich ascites tumor cells, pig thymus, and HeLa S3 cells were purified by chromatography on DNA-agarose and Blue Sepharose. A molecular mass of 112000 Da was found for all three polymerases. Fragmentation of polyacrylamide-gel-embedded polymerases with cyanogen bromide, and subsequent analysis of the fragments by polyacrylamide gradient gel electrophoresis, showed great similarities with regard to fragment sizes. The amino acid composition of the pig thymus enzyme was very similar to that of the polymerase from Ehrlich ascites tumor cells, and the terminal amino group appeared to be blocked. The HeLa polymerase electrofocused in two peaks at pH 8.8 and 5.5, while the Ehrlich ascites tumor cells and the pig thymus enzyme focused in single peaks at pH 9.4 and 9.6 respectively. Removal of residual DNA by treatment with hydroxyapatite abolished these differences in apparent isoelectric points; all three polymerases focused at pH 9.8. No important differences were found with regard to the effect of a number of substances on the synthesis of poly(ADP-ribose). Apparent Michaelis constants for NAD of 41 microM, 48 microM, and 34 microM were found for polymerase from Ehrlich ascites tumor cells, pig thymus, and HeLa S3 cells, respectively. All these results indicate that the three polymerases, which represented the major poly(ADP-ribose) polymerase activity in the organisms investigated, are closely related proteins.