The development of inhibitory antibodies against coagulation factor VIII (FVIII) in patients without congenital hemophilia A is a rare disorder known as acquired hemophilia. The etiology of the disease remains obscure. In approximately half of the patients, acquired hemophilia is associated with autoimmune disease, malignancy, multiple transfusions or the postpartum period. The remaining patients present without concomitant disorders. We hypothesized that anti-FVIII inhibitor formation might result from allo-immunization against antigenetically different FVIII variants in some patients with acquired hemophilia. The FVIII gene sequence was analyzed in 18 patients with acquired hemophilia and in two kindred of female patients with postpartal inhibitors. A hemizygous missense mutation (E2004K) was found in a male patient who developed an inhibitor after receiving multiple blood transfusions. This mutation has not been reported in the HAMSTeRS database and was not found in 50 male and 50 female healthy blood donors. It does not cause inherited hemophilia A as demonstrated by the patient’s unremarkable bleeding history before the onset of acquired hemophilia and a FVIII activity of >100 % after eradication of the inhibitor. Peptides containing E2004 or K2004 scored among the top 1 % of A3 domain-derived peptides presented on the patient’s major histocompatibility complex (MHC) class II allele, HLA-DRB1*0101. A further single amino acid substitution (D1241E) was found in four male patients and three female patients. This substitution resides in the FVIII B domain and represents a previously unknown polymorphism as it was also found in eight out of 50 male and in 17 out of 50 female controls. Comparing the allelic frequency of FVIII gene variants in patients and controls, these were equally frequent in female patients (3/18 or 17 %) and controls (18/100 or 18 %, P=0.74), but significantly more frequent in male patients (5/9 or 56 %) compared to male controls (8/50 or 16 %, P<0.01). Peptides containing D1241 or E1241 scored among the top 2 % of B domain-derived peptides presented on some MHC class II alleles (HLA-DRB1*0701 and *1501), but were unlikely to be presented on other alleles (*0101, *0301, *0401, *1101). In summary, we report allo-immunization against antigenetically relevant FVIII variants presented in a suitable MHC class II background as a novel potential mechanism of inhibitor development. Thus, exposure to ‘foreign’ FVIII variants during pregnancy or transfusion may elicit an immune response that eventually cross-reacts with ‘self’ FVIII in some patients resulting in acquired hemophilia. Similar inhibitory immune reactions may also cause loss of efficacy in patients receiving other therapeutic proteins, e.g. erythropoietin, or multiple transfusions.