The nucleotide sequence of the nonstructural protein NS1 of the influenza virus A/equine 2/Suffolk/89 was determined and found to be 97% identical to that of A/equine 2/Miami/63. A similar level of identity was shown for the deduced NS1 amino acid sequence. The NS1 gene was expressed, in its entirety and in part, as fusion proteins with glutathione S-transferase using the pGEX-3X expression vector. Antibodies to NS1 protein were detected in serum samples from ponies experimentally infected with influenza virus, but not in animals vaccinated with whole inactivated virus or in unprimed control animals. The antigenic determinant(s) of NS1 protein appear to be located in the C-terminal half of the protein. The implications of these findings are discussed with reference to the use of NS1 protein as a differential diagnostic marker for influenza virus infection in the presence of high levels of circulating antibody to influenza haemagglutinin generated by recent vaccination.