The nucleotide sequence thought to specify the glycoprotein gene, gB, of the KOS strain of herpes simplex virus type 1 (HSV-1) has been determined. A 3.1-kilobase (kb), viral-specified RNA was mapped to the left half of the BamHI-G fragment (0.345 to 0.399 map units). TATA, CAT-box, and possible mRNA start sequences characteristic of HSV-1 genes are found near 0.368 map units. The first available ATG codon is at 0.366 and the first in-phase chain terminator at 0.348 map units. A polyA-addition signal (AATAAA) occurs 17 nucleotides past the chain terminator. Translation of these sequences would yield a 100.3-kilodalton (kDa) polypeptide characterized by a 5' signal sequence, nine N-linked saccharide addition sites, a strongly hydrophobic membrane-spanning sequence, and a highly charged 3' cytoplasmic anchor sequence. Two mutants of KOS, tsJ12 and tsJ20, that are temperature-sensitive for viral growth and for the production of gB, have been physically mapped to 0.357 to 0.360 and 0.360 to 0.364 map units, respectively (DeLuca et al., in preparation). The nucleotide sequence of the mutants was determined in these regions. In both cases a single amino acid replacement within the 100.3-kDa polypeptide is predicted from the sequence analysis.