The DNA sequence was determined of the HindIII l fragment of herpes simplex virus type 2 (HSV-2), which is located in the short unique region of the HSV-2 genome. HindIII l was found to comprise 9629 base pairs. Comparison with the previously determined corresponding sequence for herpes simplex virus type 1 (HSV-1), and limited mRNA mapping, showed that HindIII l contained six genes (termed US2 to US7) and part of another (US8). The HSV-1 and HSV-2 sequences were found to be generally colinear, with one major exception: the HSV-2 DNA contained an extra sequence of about 1460 base pairs, in the coding region of gene US4. By use of an antiserum raised against an oligopeptide representing amino acids near the C terminus of the predicted HSV-2 US4 polypeptide it demonstrated that this gene encodes the virion glycoprotein gG-2, while HSV-1 US4 encodes a much smaller virion glycoprotein with homology to the C-terminal portion of gG-2. Quantitative comparisons of the HSV-2 HindIII l and corresponding HSV-1 sequences showed that they had diverged by point mutation and by local addition and deletion, as well as by the major change in genes US4. It was found that within the HSV-2-specific part of gG-2 there was a locality showing sequence similarity to a glycoprotein of pseudorabies virus (gX), and weaker similarity to glycoproteins D of HSV-1 and HSV-2. These data were interpreted to suggest, first, that HSV-2 US4 represents an ancient gene of alpha herpesviruses, and, more tentatively, that the evolution of the genes for gG and gD may have proceeded through a duplication event.