A 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A]7) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and five potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and eight potential glycosylation sites, and appears to correspond to a 90-kDa nonstructural glycoprotein (GNS) identified in BEFV-infected cells (Walker et al. [1991] J. Gen. Virol. 72, 67-74). A database search indicated that both the G and GNS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with sigma virus and vesicular stomatitis virus serotypes.