The nucleotide sequence of the NS1 gene of louping ill (LI) virus has been determined. The sequence shows a high degree of homology with other members of the tick-borne serocomplex of flaviviruses and a lower homology with the mosquito-borne flaviviruses. Alignment of the deduced NS1 amino acid sequences with all tick-borne flavivirus NS1 sequences, identified four peptide regions which were conserved for all tick-borne flaviviruses, but were variable amongst mosquito-borne flaviviruses. A dendrogram, derived from the alignment of the NS1 protein sequences, indicated an evolutionary relationship that quite closely reflects the recognised serological classification. The LI virus NS1 protein expressed in Escherichia coli and baculoviruses showed similar antigenic reactivity to the authentic virus-coded protein when tested with NS1-specific monoclonal antibodies, but did not form high molecular weight complexes and was not secreted from cells.