Previous studies [W. T. Ruyechan, L. S. Morse, D. M. Knipe, and B. Roizman (1979) J. Virol. 29, 677-697] have shown that multiple mutations are responsible for the mutant phenotypes of herpes simplex virus type 1, strain MP, and have indicated that these mutations may be located on the genome between map coordinates 0.70 and 0.83. Strain MP produces large syncytial (Syn) plaques on many cell types and does not express glycoprotein C (gC-), whereas a sibling strain mP produces wild-type, small, nonsyncytial (Syn+) plaques and is gC+. Cloned DNA fragments from strains MP and mP (and strain F) were used in marker transfer and marker rescue experiments to map more precisely the mutations in MP. It was found that a 680-bp fragment from MP DNA (map coordinates 0.735 to 0.740) could transfer a Syn marker to mP and that, conversely, an overlapping fragment from mP DNA (map coordinates 0.728 to 0.744) could rescue the Syn mutation of MP. Recombinant viruses obtained in these experiments differed from the donor of the cloned DNA fragment in plaque size, however, indicating that mutation(s) at other regions of the MP genome cause enlarged plaques, in which the infected cells are less rounded than in wild-type plaques. A fragment of MP DNA from map coordinates 0.60 to 0.64 transferred a mutation causing the gC- phenotype to strain mP, and a fragment of F DNA from map coordinates 0.62 to 0.64 rescued the gC- mutation of MP. These results, coupled with data published by Frink et al. [(1983) J. Virol. 45, 643-467], indicate that the mutation responsible for the gC- phenotype of MP may be in the structural gene for gC.