PR toxin, a mycotoxin from cultures of Penicillium roqueforti, inhibited the in vitro activities of rat liver DNA polymerase α, β, and γ irrespectively of the nature of template-primer used. The concentration required for 50% inhibition of DNA polymerase α was 5-6 × 10-6 M, while those for DNA polymerase β and γ were several times higher. By using DNA polymerase β as a model, and based on the enzyme and template-primer concentration effects and also from the kinetic analysis on PR toxin inhibition, we concluded that two action mechanisms of PR toxin inhibition on in vitro DNA synthesis are operative. Inhibition of the in vitro DNA synthesis directed by DNA template was mediated primarily through alteration of the enzyme itself, whereas in the DNA synthesis reaction directed by RNA template DNA primer, the impairment of template or primer function due to PR toxin treatment probably had occurred. The inhibition of DNA polymerase by PR toxin persisted even after exhaustive dialysis. Addition of PR toxin to an ongoing reaction also inhibited DNA synthesis. Inactivation of DNA polymerase activity of PR toxin likely involved some essential amino acid residues other than sulfhydryl groups.