Nucleotide sequences were determined for portions of the genomes of the syncytial (Syn) mutant of herpes simplex virus type 1, strain MP, and the related wild-type strain mP. Comparisons of the nucleotide sequences showed only 1 bp difference between the DNAs of strains MP and mP in the region to which the Syn mutation of MP had previously been mapped. This base pair substitution in MP (at map coordinate 0.737) eliminates a ThaI restriction endonuclease recognition site that is present in mP DNA. Analyses of MP X mP recombinant viruses showed that presence of the ThaI site correlates with the Syn+ phenotype and absence of the ThaI site correlates with the Syn phenotype as predicted. We conclude that the base pair substitution at map coordinate 0.737 is responsible for the Syn phenotype of MP. This mutation could alter translation in four of the six reading frames, causing amino acid substitutions. From only one of these reading frames is a product likely to be expressed. The 338-amino acid polypeptide that could be expressed has features characteristic of membrane-associated proteins, including hydrophobic domains, potential sites for the attachment of N-linked carbohydrate, and a potential cleavable signal sequence.