Step  Prediction and Identification of T Cell Epitopes in the H5N1 Influenza Virus Nucleoprotein in Chicken T cell epitopes can be used for the accurate monitoring of avian influenza virus (AIV) immune responses and the rational design of vaccines. No T cell epitopes have been previously identified in the H5N1 AIV virus nucleoprotein (NP) in chickens. For the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken MHC class Ι molecules for four commonly encountered unique haplotypes, i.e., B4, B12, B15, and B19. H5N1 AIV NP was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of MHC class I molecules of the B4, B12, B15 and B19 haplotypes. Seventy-five peptide sequences were modelled and their MHC class I molecule-binding abilities were analysed by molecular docking. Twenty-five peptides (Ten for B4, six for B12, two for B15, and seven for B19) were predicted to be potential T cell epitopes in chicken. Nine of these peptides and one unrelated peptide were manually synthesized and their T cell responses were tested in vitro. Spleen lymphocytes were collected from SPF chickens that had been immunised with a NP-expression plasmid, pCAGGS-NP, and they were stimulated using the synthesized peptides. The secretion of chicken IFN-γ and the proliferation of CD8(+) T cells were tested using an ELISA kit and flow cytometry, respectively. The significant secretion of chicken IFN-γ and proliferation of CD8(+) T lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides NP(89–97) and NP(198–206), respectively. The results indicate that peptides NP(89–97) (PKKTGGPIY) and NP(198–206) (KRGINDRNF) are NP T cell epitopes in chicken of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chicken. The introduction into the human population of animal-derived influenza A viruses with a novel haemagglutinin (HA), or a novel HA and neuraminidase (NA), and their subsequent spread could result in global influenza pandemics [1] . Since 2003, the highly pathogenic H5N1 avian influenza virus (AIV) has caused numerous cases of severe disease and death in humans [2] . An influenza pandemic could ensue if this virus developed the capacity to spread easily among humans [3] [4] [5] . Migratory birds constitute the natural reservoir for AIVs, but chickens may play a key role in the transmission to humans [6] . Epitopes can be used for accurately monitoring immune responses to AIV and for the rational design of protective vaccines. However, only two epitopes from the H5N1 avian influenza A/Vietnam/1194/2004 virus are included in the Immune Epitope Database and Analysis Resources (IEDB). The majority of T cell and B cell epitopes have been identified in mouse, human, or rabbit hosts. Few epitopes have been described in chicken [7] .