A polysaccharide was purified from the slime of a myxomycete, Physarum polycephalum, and its inhibitory effect on eukaryotic DNA polymerases was examined. Almost all the calf thymus DNA polymerase alpha activity was inhibited with higher than 0.2 mg/ml of the polysaccharide, when the assay was carried out with activated DNA as a template. The inhibitory effect occurred regardless of the amounts of the enzyme and deoxyribonucleotides, however, kinetic analysis revealed that the inhibition occurs competitively with the template DNA, the Ki value being 4 micrograms/ml. Inhibition was observed for DNA polymerase alpha, but not for DNA polymerases beta and gamma from various eukaryote species.