Heat-inactivated FVIII has reduced immunogenicity. FVIII^−/−/C57BL/6 mice received 5 intraperitoneal injections of either 1 μg native-FVIII (n = 7) or 1 μg heat-inactivated FVIII (n = 7) and were bled 7 days after the last injection. This experiment was repeated at least 4 times, and representative results are reported here. (A) One week after the last injection, mice were bled and serum samples were analyzed by ELISA. Antibody concentrations were calculated based on standard curves using monoclonal antibodies of known concentration. Native FVIII is highly immunogenic with mean antibody concentrations of 495 ± 131 μg/mL, whereas the inactivated form is less immunogenic at 136 ± 41 μg/mL (*P = .011). (B) Splenocytes were processed into a single-cell suspension and assayed for ³H incorporation as previously described. T cells from native FVIII-treated mice proliferated in response to FVIII, whereas the recall response from mice immunized with heated FVIII was significantly lower (*P = .015).