We have mapped the location in herpes simplex virus (HSV) DNA of (i) three mutations at different loci ( syn loci) which alter the social behavior of infected cells from clumping of rounded cells to polykaryocytosis, (ii) a mutation which determines the accumulation of one major glycoprotein [VP8.0(C 2 )], and (iii) the sequences encoding four major virus glycoproteins [VP8.0(C 2 ), VP7(B 2 ), VP8.5(A), and VP19E(D 2 )]. The experimental design and results were as follows. (i) Analysis of HSV-1 × HSV-2 recombinants showed that the sequences encoding the VP19E(D 2 ) glycoprotein map in the S component, whereas the sequences encoding the other three major glycoproteins are in two locations in the L component of HSV DNA. The templates specifying the HSV-1 and HSV-2 glycoprotein VP8.0(C 2 ) appear not to be colinear; we isolated recombinants specifying glycoproteins comigrating in sodium dodecyl sulfate-polyacrylamide gels with VP8.0(C 2 ) of both HSV-1 and HSV-2. (ii) Marker rescue of a ts mutant defective in accumulation of glycoprotein VP7(B 2 ) showed that the mutation maps within a region containing the sequences encoding that glycoprotein. (iii) Marker transfer experiments involving transfection of rabbit skin cells with donor HSV-1(F) DNA and fragments from several donor strains causing fusion of Vero or both Vero and HEp-2 cells revealed the existence of three syn loci specifying the social behavior of cells and one locus ( Cr ) determining the accumulation of glycoprotein VP8.0(C 2 ). The Cr locus maps to the right of the template specifying VP8.0(C 2 ) glycoprotein. Loci syn 1 and syn 2 map at or near the Cr locus but can be segregated from it. Locus syn 3 maps at or near the template specifying glycoproteins VP7(B 2 ) and VP8.5(A). The expression of mutations in the syn 1 and syn 3 loci appear to be cell type dependent, in that recombinants with these mutations fuse Vero cells but not HEp-2 cells. Recipients of the syn 2 locus or of both syn 2 and syn 1 loci fuse both Vero and HEp-2 cells.