An antibody made against the herpes simplex virus 1 US5 gene predicted to encode glycoprotein J was found to react strongly with two proteins, one with an apparent Mr of 23,000 and mapping in the S component and one with a herpes simplex virus protein with an apparent Mr of 43,000. The antibody also reacted with herpes simplex virus type 2 proteins forming several bands with apparent Mrs ranging from 43,000 to 50,000. Mapping studies based on intertypic recombinants, analyses of deletion mutants, and ultimately, reaction of the antibody with a chimeric protein expressed by in-frame fusion of the glutathione S-transferase gene to an open reading frame antisense to the gene encoding glycoprotein B led to the definitive identification of the new open reading frame, designated UL27.5. Sequence analyses indicate the conservation of a short amino acid sequence common to US5 and UL27.5. The coding sequence of the herpes simplex virus UL27.5 open reading frame is strongly homologous to the sequence encoding the carboxyl terminus of the herpes simplex virus 2 UL27.5 sequence. However, both open reading frames could encode proteins predicted to be significantly larger than the mature UL27.5 proteins accumulating in the infected cells, indicating that these are either processed posttranslationally or synthesized from alternate, nonmethionine-initiating codons. The UL27.5 gene expression is blocked by phosphonoacetate, indicating that it is a gamma2 gene. The product accumulated predominantly in the cytoplasm. UL27.5 is the third open reading frame found to map totally antisense to another gene and suggests that additional genes mapping antisense to known genes may exist.