A 3914 nucleotide region of the Adelaide River virus (ARV) genome, located immediately downstream of the M2 gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs). The first encodes a protein comprising 660 amino acids which shares extensive sequence homology with the virion G protein of bovine ephemeral fever virus (BEFV) and less but significant homology with other rhabdovirus glycoproteins. The size and structural characteristics of the product indicate that it represents the 90-kDa ARV virion G protein. The second ORF encodes a polypeptide of 609 residues with nine potential glycosylation sites which is most closely related to the BEFV non-structural glycoprotein (GNS). In infected mammalian cells, the ARV G and GNS genes are transcribed primarily as a polycistronic mRNA which appears to extend from the consensus sequence (AACAG) at the start of the G gene to the next recognized polyadenylation signal (CATG[A]7) located 697 nucleotides downstream of the GNS protein termination codon. Less abundant mRNAs which appeared to initiate at consensus sequences immediately preceding and following the GNS ORF and terminate at the same polyadenylation signal were also detected. Polyadenylation-like sequences at the end of each ORF do not appear to be recognized as transcription stop signals. Multiple sequence alignments and phylogenetic analyses indicated that the ARV G and GNS glycoproteins, like those of BEFV, are structurally related and appear to have evolved at different rates from a common ancestral gene. A copy-choice mechanism, involving upstream relocation of the polymerase during replication, is proposed to account for the evolution of the tandem glycoprotein genes.