Mild/moderate hemophilia A patients carrying certain mutations in the C1 domain of factor VIII (FVIII) have a higher risk of inhibitor occurrence. To analyze the mechanisms responsible for inhibitor development in such patients, we characterized FVIII-specific CD4(+) T-cell clones derived from a mild hemophilia A patient carrying an Arg2150His substitution in the C1 domain and who presented with a high titer inhibitor toward normal but not self-FVIII. All T-cell clones recognized synthetic peptides encompassing Arg2150. The peptides were presented to the T-cell clones by DRB1*0401/DRB4*01 or DRB1*1501/DRB5*01. Interestingly, the latter haplotype was previously reported as being associated with an increased incidence of inhibitor formation. Peptide I2144-T2161 also bound to other DR molecules such as DRB1*0101 and DRB1*0701, indicating that the peptide binds to major histocompatibility complex (MHC) class II molecules expressed in more than 60% of the population. None of the T-cell clones recognized recombinant FVIII carrying the substitution Arg2150His, even when FVIII was presented by an FVIII-specific B-cell line. The mutation likely alters T-cell recognition of the mutated peptide associated to MHC molecules, because the mutated peptide bound to immunopurified DR molecules nearly as effectively as the native peptide. These observations demonstrate that T cells of this patient with mutation Arg2150His distinguish between self- and wild-type FVIII and provide a plausible mechanism for the frequent occurrence of an inhibitor in patients carrying this substitution. A similar phenomenon may occur with other mutations associated to an increased incidence of inhibitor formation.