Quantification of inhibitory antibodies against infused factor VIII (FVIII) has an important role in the management of patients with hemophilia A. This article summarizes results from the largest North American FVIII inhibitor proficiency testing challenge conducted to date. Test samples, 4 negative and 4 positive (1-3 Bethesda units [BU]/mL), were distributed by the ECAT Foundation in conjunction with the North American Specialized Coagulation Laboratory Association and analyzed by 38 to 42 laboratories in 2006 and 2007. Whereas laboratories were able to distinguish between the absence and presence of low-titer FVIII inhibitors, the intralaboratory coefficient of variation was high (30%-42%) for inhibitor-positive samples, and the definition of lower detection limits of the assay was variable (0-1 BU/mL). Most laboratories performed the Bethesda assay with commercially supplied buffered normal pooled plasma in a 1:1 mix with patient plasma. These data provide information for the development of consensus guidelines to improve FVIII inhibitor quantification.