The ability of three thermostable enzymes, Tth, Taq, and Klentaq DNA polymerases, to amplify DNA with primers containing mismatches in the 3'-terminal region was studied. It is shown that Tth polymerase, in contrast to the Taq and Klentaq enzymes, synthesizes equally well DNA with primers perfectly complementary to the template and with those containing mismatches next the 3'-end. The use of Tth DNA polymerase in the polymerase chain reaction was shown to result, in some cases, in a great number of additional, nonspecific DNA fragments as compared with Taq DNA polymerase. This may be due to the ability of Tth polymerase for DNA primer extension even if the 3'-terminal region of the primer contains nucleotides non-complementary to the template. Tth DNA polymerase and a Klentaq/Tth mixture (100:1) can be efficiently used in the amplification of DNA with degenerated primers and primers forming nonperfect duplexes with the template.