In an attempt to assess the importance of the nucleoprotein (NP) in the determination of host specificity, a series of experiments was performed on influenza A viruses of the H3N2 subtype. We have examined rescue of mutants of A/FPV/Rostock/34 with temperature-sensitive (ts) lesions in the nucleoprotein (NP) gene by double infection of chick embryo cells with H3N2 strains isolated from different species. The ts mutants could be rescued by all avian H3N2 strains but not by any of the human H3N2 isolates. Only two of the swine H3N2 strains tested were able to rescue our mutants. The NP gene of these two swine isolates resembled the NP gene of the avian strains genetically in the hybridization test. However, their NPs reacted differently with a set of monoclonal antibodies when compared with NPs of avian H3N2 strains. Concerning multiplication in ducks they behaved like the other swine and human strains. The phosphopeptide fingerprints of all swine isolates tested were alike and were different from those of human or avian origin. Our observations are compatible with the idea that human H3N2 strains might not be able to cross the species barrier to birds directly, and possibly also not the other way around, without prior reassortment in pigs, which seem to have a broader host range concerning the compatibility of the NP gene in reassortants.