Five herpes simplex virus mutants containing temperature-sensitive mutations in the gene for the major DNA-binding protein were assayed for their sensitivities to the DNA polymerase inhibitors aphidicolin and phosphonoacetic acid (PAA). Four of the mutants (tsA1, tsA15, tsA24, and tsA42) exhibited altered sensitivity to one or both of the inhibitors relative to the wild-type parent. In tsA1, a mutation or mutations conferring aphidicolin and PAA hypersensitivity were mapped by corescue with the temperature-sensitivity marker of tsA1 to a region of the DNA-binding protein locus, between map coordinates 0.385 and 0.398. The mutation conferring PAA hypersensitivity in tsA24 similarly corescued with the tsA24 temperature-sensitivity marker, mapping to the DNA-binding protein locus between coordinates 0.398 and 0.413. Thus, mutations outside the DNA polymerase locus and within the DNA-binding protein locus can confer altered sensitivity to certain DNA polymerase inhibitors. Assays of the aphidicolin and PAA sensitivities of ts+ recombinants derived by marker rescue of the DNA-binding protein mutants revealed the presence of additional mutations, separable from the ts mutations, in each of three mutants examined. One such mutation, which contributed to the aphidicolin-hypersensitivity phenotype of tsA1, mapped between coordinates 0.422 and 0.448, and resides, most probably, within the DNA polymerase locus. These additional mutations possibly confer compensating modifications to the DNA polymerase such that functional interaction with altered DNA-binding protein is restored. These findings provide strong evidence that the major DNA-binding protein and the DNA polymerase of herpes simplex virus interact in infected cells.