Abstract Fine structure physical map locations were determined for syncytial mutants (MP, syn-20, syn-102, syn-103, and syn-105) of Herpes Simplex Virus type 1(HSV-1). All except MP were derived from the KOS strain. MP contains multiple mutations, including one that leads to the loss of accumulation of glycoprotein gC ( Ruyechan, 1979). Overlapping DNA subclones within the prototypic map coordinates 0.707 to 0.810 were constructed from a library of KOS fragments. These were used along with intact mutant DNA to rescue the syn marker. Mutations in all of the mutants were rescued by KOS DNA sequences between 0.732 and 0.745. This cell-dependent syn mutation is the only lesion in the KOS-derived mutants. A second syn mutation in MP was mapped at coordinates 0.745 to 0.753. This lesion produces less fusion and is also cell-type dependent for the fusion phenotype. Cell-type independent fusion requires the presence of both mutations. The locus determining glycoprotein C (gC) production in strain MP was also mapped, using indirect immunofluorescence, to coordinates 0.745 to 0.753. Nucleotide sequences for ICP-27, an immediate early or a protein of unknown function, are within these coordinates. Since gC production and the syn phenotype are separable by recombination, they must be caused by independent mutations.