A intertypic recombinant, designated HSV-R(D1), had previously been generated from non-neuroinvasive HSV-2(186) and neuroinvasive HSV-1(17). Although the recombinant contained<2% of the HSV-1 genome, it retained the neuroinvasive phenotype. The nucleotide sequences responsible for the neuroinvasiveness of HSV-R(D1) were previously mapped to a 3.0 kb segment of DNA located within the DNA polymerase gene (mu 0.414 to 0.430) via marker rescue experiments. We have now sequenced this region and compared our results to the published nucleotide sequence of the HSV-1 (17) and HSV-2(186) DNA polymerase genes. It was found that the 3.0 kb HSV-R(D1) DNA fragment consisted entirely of HSV-2(186) nucleotide sequences except for the presence of 1610 bp of HSV-1 (17) DNA. The 1610 bp of HSV-1 DNA coded for a 536 amino acid (AA) region which were located between AA 254 and 790 of the DNA polymerase enzyme. Comparison of the 536 AA sequence of neuroinvasive HSV-1 (17) with the homologous area of the non-neuroinvasive HSV-2(186) DNA polymerase indicated that the two polymerases differed at 56 AA positions. In addition, this area of the HSV-1 (17) DNA polymerase was 5 AA acids shorter than the HSV-2(186) DNA polymerase. Specific amino acid changes that might account for the neuroinvasive phenotype of HSV-R(D1) are discussed.