Abstract Autoreactivity may play a critical role in the chronification of atopic dermatitis (AD). Several studies showed that AD patients produce IgE Abs specific for autoantigens, and we described Th as well as CD8+ T cells specific for the autoallergen Hom s 2, the α-chain of the nascent polypeptide-associated complex (α-NAC). This study aimed to investigate the frequency and inflammatory phenotype of autoallergen-specific CD8+ T cells. CD8+ T cell immunodominant epitopes of α-NAC were mapped by applying prediction softwares, and binding affinity was confirmed by stabilization of empty MHC complexes. MHC class I tetramers were assembled and binding cells were analyzed directly ex vivo by flow cytometry and in terms of single-cell assessment by ChipCytometry. We report significantly elevated numbers of α-NAC–specific peripheral T cells in sensitized patients compared with nonatopic controls. These cells secrete IL-4 and IFN-γ, and surface markers revealed significantly elevated frequencies of circulating terminally differentiated α-NAC–specific CD8+ T cells in patients with AD compared with nonatopic donors. The observed phenotype of α-NAC–specific CD8+ T cells indicates a role in the pathogenesis of AD.