Nucleotide sequence analysis of a 42-kb region of the vaccinia virus (strain Western Reserve) genome identified a gene with the potential to encode a 35.1-kDa polypeptide with properties of a membrane glycoprotein (Smith et al., J. Gen. Virol. 72, 1349-1376, 1991). The 317 amino acid open reading frame (ORF) has similarity with complement control proteins and a secretory vaccinia virus protein (C28K) which interferes with complement function. The predicted B5R gene product differs from the latter protein in that it contains a C-terminal hydrophobic sequence and may be membrane-associated rather than secretory. Transcriptional mapping by Northern blotting and S1 nuclease protection showed that the gene is transcribed both early and late during infection, with the early RNA start site located 60 bp upstream of the late start site that is present at -9 to -5 bp relative to the ORF. Nevertheless, translation of early and late mRNAs are predicted to produce the same polypeptide. A rabbit antiserum was raised to the predicted external hydrophilic domain of B5R expressed in Escherichia coli and used to immunoprecipitate a M(r) 42 K protein from vaccinia-infected cells. This protein was synthesized throughout infection, with a peak from 6 to 7 hr, and its production was inhibited by tunicamycin but not monensin. Western blotting of proteins from purified extracellular enveloped virus (EEV) or intracellular naked virus with anti-B5R serum showed that this M(r) 42 K protein and two higher molecular weight forms (Mr82 and 87 K) were present only in EEV. Anti-B5R serum inhibited comet formation by the IHD-J strain of virus on RK13 cells. B5R is the third vaccinia gene shown to encode an EEV glycoprotein, the others being the virus hemagglutinin gene, and gene SalL4R which encodes a group of lectin-like glycoproteins of M(r) 22-24 K.