Inhibitory effects of 2',3'-dideoxyribonucleoside 5'-triphosphates (ddNTP's) on the activity of mouse myeloma DNA polymerase alpha were examined. In contrast to the widely accepted conclusion that DNA polymerase alpha was not inhibited by ddNTP, our results showed that all 4 ddNTP's (ddATP, ddCTP, ddGTP, ddTTP) exhibited strong inhibitory power to this enzyme in the presence of Mn2+. The observed inhibitions by ddNTP's were neither due to chain termination of growing DNA nor to enzyme inactivation by these compounds, but due to competition with deoxynucleoside triphosphate with the same base. The inhibition constant (Ki) varied depending on the combination of template-primer, substrate and inhibitor. The results suggest that Mn2+ plays a role in increasing affinity of the dideoxynucleotides to DNA polymerase alpha by interacting with anyone or more of the dideoxynucleotide, enzyme and template-primer.