In continuation of efforts to correlate the antitemplate activities of chemically modified polynucleotides with their base composition and structure, four synthetic copolymers, poly(A,C), poly(C,U), poly(A,C,U), and poly(A,C,G) were modified by thiolation of 2.6-4.8% of their pyrimidine bases. The resulting 5-mercaptoheteropolynucleotides and the previously described 5-mercapto-polycytidylate (MPC) and -polyuridylate (MPU) were tested in a comparative manner as inhibitors of the DNA polymerase-alpha from rat hepatoma. A wide scale of inhibitory potencies was obtained in the following (decreasing) order: MPU greater than M-poly(A,C,U) greater than M-poly(C,U) greater than MPC greater than M-poly(A,C) greater than or equal to M-poly(A,C,G). The sensitivity of the hepatoma DNA polymerase toward these antitemplates increased upon further purification of the enzyme through the DNA-agarose step. Partially thiolated DNA-isolates from rat hepatoma and calf thymus, respectively, showed significant inhibition of the hepatoma DNA polymerase, the thiolated hepatoma DNA being the more active inhibitor.