A 2341-nucleotide region of the Adelaide River virus (ARV) genome, located immediately downstream of the second glycoprotein (GNS) gene, has been cloned and sequenced. The region contains four long open reading frames (ORFs), the last of which represents a 1088-nucleotide fragment at the start of the ARV L gene. Between the GNS and L genes are two coding regions, separated by a single nucleotide (C), and each bounded by recognized transcription initiation (AACAG) and termination/polyadenylation (CATG[A]7) sequences. The first coding region comprises 682 nucleotides and contains two long ORFs (alpha 1 and alpha 2) which are in the same reading frame but separated by two consecutive stop codons. The alpha 1 ORF encodes a 12,545-Da polypeptide which contains highly hydrophobic and highly basic domains. The alpha 2 ORF includes a potential initiation codon 18 nucleotides downstream of the tandem stop codons and encodes a polypeptide of 11,951 Da. In ARV-infected cells, the alpha region is transcribed primarily as a long 4.7-kb polycistronic mRNA containing the G, GNS, alpha 1, and alpha ORFs. Direct sequence analysis of the mRNA indicated that the tandem stop codons between the alpha 1 and alpha 2 ORFs are retained in the transcript. The second coding region contains a single long ORF (beta) comprising 493 nucleotides which encodes a polypeptide with a calculated pl of 6.614 and molecular weight of 17,102 Da. The putative beta protein is similar in size to a protein which has been reported as a minor component of virions. The beta gene is transcribed as a 0.65-kb monocistronic mRNA for which the putative transcription termination/polyadenylation signal overlaps the L gene by 22 nucleotides.