A gene designated SalF5R from within the SalI F fragment of vaccinia virus strain WR has been characterized. The predicted primary translation product has 194 amino acids with an Mr of 22578 and has features typical of a class I membrane glycoprotein. At the amino and carboxy termini there are runs of hydrophobic residues that might function as membrane signal and anchor sequences, respectively, and after the C-terminal hydrophobic sequence there is a short charged sequence that may prevent passage of the molecule through the membrane. Between these hydrophobic regions there are two potential sites for addition of N-linked carbohydrate. Northern blotting using a probe to an internal region of the open reading frame (ORF) detected an early transcript of 750 nucleotides and late transcripts of heterogeneous length. However, accurate mapping of the 5' ends of these transcripts did not reveal a monocistronic mRNA that might allow translation of the full-length SalF5R ORF. Instead two early transcripts were found, one beginning 130 nucleotides downstream of the start of the ORF, and a second initiating in the short intergenic region between SalF5R and SalF6R. The nearest late initiation site mapped to the beginning of the upstream gene SalF4R (profilin) and not the beginning of the SalF5R ORF. A virus deletion mutant lacking an internal 554 nucleotides of the 582 bp SalF5R ORF was constructed by insertion of the Escherichia coli guanine phosphoribosyl transferase gene linked to the vaccinia virus p7.5K promoter. The rate of replication and the final titre of intracellular naked virus produced by this virus was indistinguishable from wild-type virus in CV-1 and RK13 cells. In intranasally infected mice the virus lacking SalF5R was not attenuated in comparison to wild-type virus. The SalF5R site may be useful for insertion of foreign DNA into recombinant vaccinia viruses.