The contribution of influenza A neuraminidase antibodies to the reaction with whole virus in an enzyme-linked immunosorbent assay (ELISA) was assessed by specific absorption of rabbit hyperimmune sera. Although measurable and independent, the effect of neuraminidase antibodies was less than that of hemagglutinin antibodies. Recombinants with an irrelevant hemagglutinin were used successfully as antigens in an ELISA test for measuring neuraminidase antibodies in rabbit hyperimmune sera, but a low cross-reaction between N1 and N2 subtypes was observed. However, for the measurement of N2 antibody rises in human sera, ELISA was highly specific and compared favorably with two other methods, neuraminidase inhibition and single radial hemolysis.