A characterization of vaccinia virus strain Western Reserve (WR) open reading frame (ORF) A36R is described. This ORF is predicted to encode a 221-amino-acid protein (M(r) 25.1 K) with an amino-terminal hydrophobic sequence, seven potential sites for attachment of N-linked carbohydrate, but no carboxy-terminal transmembrane anchor. It is identical in vaccinia strain Copenhagen and shares 94.6% amino acid identity with the corresponding ORF in variola virus strains Harvey, India-1967, and Bangladesh-1975. RNA analyses detected a 600-nucleotide, early transcript that initiated 10-13 nucleotides upstream of the A36R ORF, and heterogeneously sized late transcripts running across the ORF. A rabbit antiserum raised against an Escherichia coli glutathione S-transferase fusion protein identified M(r) 43-50 K proteins that accumulated late during vaccinia virus infection and fractionated as integral membrane proteins during Triton X-114 partitioning. Similar polypeptides were expressed by vaccinia virus strains Tian Tan, Tashkent, Lister, Wyeth, Copenhagen, and IHD-J and by rabbitpox virus and cowpox virus (strain Brighton Red). Immunoblot analysis of purified and protease-digested intracellular mature virus (IMV) and extracellular enveloped virus (EEV) showed that the A36R proteins were present on the surface of EEV with type II membrane topology, but were absent from IMV. A WR deletion mutant lacking the A36R ORF (delta A36R) had a small plaque phenotype on all cell lines tested. IMV formation by delta A36R was unaltered but EEV formation was reduced approximately fivefold compared to wild-type (WT) when measured either by density gradient analysis of isotopically labeled virions or by infectivity assays. Thus the loss of the A36R protein from the EEV surface did not reduce EEV specific infectivity in vitro. Despite this, delta A36R showed striking attenuation compared with WT in a murine intranasal model. Finally, a revertant virus in which the A36R ORF was restored showed WT plaque size, EEV formation, and virulence, demonstrating that all the phenotypic differences of delta A36R were attributable to loss of the A36R gene and not to other mutations acquired during its construction.