The sequence of the sense strand of RNA segment 5 of both Australian and South African bluetongue virus (BTV) serotype 1 has been determined and found to be 1771 and 1773 nucleotides in length, respectively. Both coding sequences of 1656 nucleotides were flanked by a 5' non-coding sequence of 34 nucleotides and 3' non-coding regions of 78 and 80 nucleotides, respectively. The methionine codons at residues 35-37 were assumed to initiate the synthesis of 64.6 or 64.415 kDa proteins which had calculated net charges of +5 or +4 at neutral pH, respectively. The encoded NS1 proteins had a very high molar ratio of cysteine residues. A variable region of approximately 45 nucleotides at the 3'-terminus of RNA segment 5 of South African and Australian BTV-1 and the RNA segment 6 of the North American BTV-10 was shown to be unusually rich in A + T residues (approximately 80-82%) compared with other BTV gene segments so far sequenced which have between 52 and 56% A + T. These regions were thought to be responsible for the variable migration of RNA 5 segments on electrophoresis in polyacrylamide gels in the presence of urea. This variability in the apparent molecular weight of RNA 5 segments was not restricted to BTV amongst Australian orbiviruses tested, nor was the apparent molecular weight for RNA 5 identical for different isolates of the same BTV serotype, indicating that this A + T rich region was highly variable. Comparison of the nucleotide and amino acid sequence divergence of the Australian and South African BTV RNA segments 5 to that for the North American BTV-10 RNA segment 6 (which codes for NS1) revealed the same relationships as those found for the core protein VP3 gene sequences, in that although all NS1 proteins were very similar in their amino acid sequences, their genes were more variable. The Australian NS1 sequence differed from both the South African and North American genes by 20% at the nucleotide level, whereas the North American and South African sequences diverged by only 11%. Hybridization analyses showed that RNA segment 5 DNA probes were capable of delineating the geographical origin of a BTV isolate, as had been observed for VP3 probes; however, other probes were also generated which were capable of unambiguously differentiating BTV isolates from other orbiviruses tested.