A double antibody sandwich blocking ELISA, using a monoclonal antibody (MAb) against influenza A nucleoprotein (NP) was developed to detect antibodies against influenza. Collections of serum samples were obtained from human and various animal species. All influenza A subtypes induced antibodies against hemagglutinins and NP. A close correlation between titers of the hemagglutination inhibition (HI) test and the NP-ELISA was seen. Antibodies against influenza NP were demonstrated in serum samples from humans, ferrets, swine, horses, chickens, ducks, guinea pigs, mice, and seals. The serum samples were collected at intervals during prospective epidemiological studies, from experimental and natural infections, and vaccination studies. The decline of maternal antibodies was studied in swine and horses. The NP-ELISA enables rapid serological diagnosis and is suited for influenza A antibody screening, especially in species which harbor several influenza subtypes. The HI and neuraminidase inhibition tests, however, must still be used for subtyping.