The nucleotide sequence (2041 nucleotides) of a genomic region of herpes simplex virus type 1 (KOS strain) associated with virus-induced cell fusion has been determined. The sequence is bounded by a NruI site at 0.732 and a BamHI site at 0.745 prototypic map units. An open reading frame in the left-to-right orientation specifies a protein of 338 amino acids. The protein is positively charged. Since secondary structure analysis predicts four extensive hydrophobic domains the protein is probably a membrane-associated or a transmembrane protein. Transcription of the putative fusion gene is dependent on viral DNA synthesis, characteristic of the late (gamma) viral gene class. Two syncytia-inducing mutations, syn20 and MP, have been previously mapped to a 504-base pair PstI fragment within these genomic coordinates (V. C. Bond and S. Person (1984), Virology 132, 368-376). The nucleotide sequence of the PstI fragment was determined for the two mutants. Both were shown to have an amino acid substitution at residue 40 of the fusion protein. A second change at residue 101 for MP is probably unrelated to the fusion phenotype.