In an earlier paper (Morse et al., J. Virol 24:231--248, 1977) we reported on the provenance of the DNA sequences in 26 herpes simplex virus type 1 (HSV-1) X HSV-2 recombinants as determined from analyses of their DNAs with at least five restriction endonucleases. This report deals with the polypeptides specified by the recombinants and by their HSV-1 and HSV-2 parents. We have identified (i) the corresponding HSV-1 and HSV-2 polypeptides with molecular weights ranging from 20,000 to more than 200,000, (ii) the polypeptides that undergo rapid post-translational processing, and (iii) polypeptides that vary intratypically in apparent molecular weight. By comparing the segregation patterns of the polypeptides with those of the DNA sequence of the recombinants, we have mapped the templates specifying 26 polypeptides and several viral functions on the physical map of HSV DNA. The data show the following: (i) alpha polypeptides map at the termini of the L and S components of the HSV DNA. Although alpha ICP 27 maps entirely within the reiterated region of the L component, the template for alpha ICP 4 may lie only in part within the reiterated sequences of the S component. Of note is the finding that cells infected with a recombinant that contains both HSV-1 and HSV-2 DNA sequences in the S component produced alpha ICP 4 of both HSV-1 and HSV-2. (ii) Templates specifying beta and gamma polypeptides map in the L component and appear to be randomly distributed. (iii) Thymidine kinase and resistance to phosphonoacetic acid mapped in the L component. In addition, we have taken advantage of the rapid inhibition of host protein synthesis characteristic of HSV-2 infections and syncytial plaque morphology to also map the template(s) responsible for these functions in the L component. The implications of the template arrangement in HSV DNA are discussed.