Almost all influenza virus proteins are found to contain caspase cleavage motifs. Two caspase cleavage consensus sequences, EXD|Y and D/EXXD|Y (caspase motifs) were identified in N- and C-terminal regions of influenza virus proteins nucleocapsid NP (positions D 16 and D 497) and ionic channel M2 (positions D 23 and D 87). Using reverse genetics with the highly-virulent avian influenza virus A/FPV/Rostock (H7N1), as a vector precursor, these NP and M2 caspase motifs were artificially altered by site-directed mutagenesis and pathogenicity of the generated caspase mutant viruses was tested in chickens. Three main groups of virus mutants were identified. The first group of mutants was characterized by high replication in cells and low virulence in chickens. These virus mutants possessed the altered N-terminal NP and C-terminal M2 caspase motifs. The second group of virus mutants, possessing the altered N-terminal caspase motif of M2, was characterized by attenuated replication in cultured cells and reduced pathogenic properties in chickens. Third, mutations generated in the C-terminus of NP were lethal and restricted virus rescue by reverse genetics, implying a critical role of this caspase site in virus replication. Thus, these data suggested that, (i) caspase motifs in virus proteins play a significant role in virus pathogenicity; (ii) the lack of direct correlation between replication potential and pathogenicity, observed in caspase mutants of the first virus group, implied that virus caspase motifs could affect immunopathogenesis during the infection process, rather than simply controlling virus production in target cells in the chicken host.