The objective of this study was to predict and verify T cell epitopes in the H5N1 AIV NP in chicken. Using a motif combined with a structure-based method, 25 potential T cell epitope peptides were predicted in the H5N1 AIV NP in chickens of B4, B12, B15, and B19 haplotypes. NP 89-97 and NP 198-206 were found to induce a significant proliferation of CD8 + T lymphocytes and they increased the secretion of chicken IFN-c in sensitized splenic lymphocytes. These data suggest that peptides NP 89-97 and NP 198-206 are NP T cell epitopes in chickens of certain haplotypes. This study is important for the following two reasons. First, this is the first study to determine the structural characteristics of the peptide-binding domains of chicken MHC class I molecules belonging to the B4, B12, B15, and B19 haplotypes using a combined motif-structure method to predict T cell epitopes in chickens. Second, NP 89-97 and NP 198-206 are the first two T cell epitopes to be identified in AIV NP in chickens of certain haplotypes.