Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor α (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer⁺/TRAJ24⁺/CD4⁺ T cells in DQ6⁺ individuals with and without narcolepsy. We identify related TRAJ24⁺ TCRαβ clonotypes encoded by identical α/β gene regions from two patients and two controls. TRAJ24-G allele⁺ clonotypes only expand in the two patients, whereas a TRAJ24-C allele⁺ clonotype expands in a control. A representative tetramer⁺/G-allele⁺ TCR shows signaling reactivity to the epitope HCRT_87–97. Clonally expanded G-allele⁺ T cells exhibit an unconventional effector phenotype. Our analysis of in vivo expansion of HCRT-reactive TRAJ24⁺ cells opens an avenue for further investigation of the autoimmune contribution to narcolepsy development.