6-(p-n-Butylanilino)uracil and N2-(p-butylphenyl)guanine inhibited the activity of DNA polymerase alpha from calf thymus but had no effect on other eukaryotic polymerases (DNA polymerases beta and gamma) or Escherichia coli DNA polymerase I. Inhibition was competitive with deoxyguanosine 5'-triphosphate and did not occur in the reaction of DNA polymerase alpha with a template that did not contain cytosine residues. The results support a mechanism which involves hydrogen bonding of inhibitors with cytosines in the DNA template and binding with an inhibitor specific site on the enzyme. A screen of inhibitor effects on normal and cancer cell growth in culture showed that cells were not uniformly sensitive to these compounds, a mouse lymphoma line being least sensitive and a human lung cancer line being most sensitive. It is suggested that these inhibitors may be useful to probe possible structural differences among DNA polymerases alpha.