The interaction of 6-(phenylhydrazino)-pyrimidines and Bacillus subtilis DNA polymerase III was examined in experiments exploiting agarose gel filtration of mixtures of drug, DNA, and purified enzyme. 6-(p-Hdroxyphenylhydrazino)-uracil and 6-(p-hydroxyphenylhydrazino)-isocytosine were used as model inhibitors; both drugs induced the formation of a distinct polymerase-DNA complex. Comples formation required the inhibitory, hydrazino forms of the drugs and a form of DNA suitable as a primer-template for DNA polymerase III. dGTP and dATP, which respectively, competitively antagonize the inhibitory effects of the uracil and isocytosine derivatives, antagonized in an equally specific manner the respective capacities of these compounds to induce complex formation. Experiments exploiting both wild type and drug-resistant, mutant polymerases indicated that drug concentrations required for the half-maximal induction of complex formation were nearly identical with the apparent inhibitor constants (Ki) determined independently by kinetic analysis of enzyme inhibition. These results and those of experiments exploiting defined homopolymer-oligomer combinations as template-primers support a model of inhibitor action in which arylhydrazinopyrimidine forms a reversible, ternary complex with the enzyme and an appropriate timplate pyrimidine residue in an area adjacent to the 3-hydroxyl primer terminus.