The factor VIII activity of B-domain deleted recombinant factor VIII (BDDrFVIII) measured by activated partial thromboplastin time (APTT)-based one-stage assays is approximately 50% of the activity obtained by the chromogenic assay. Similar results have been reported for the two licensed full-length recombinant factor VIII products. In view of these findings, comprehensive studies have been undertaken to find the cause of the assay differences. Only the phospholipid reagent, used as a platelet substitute in the one-stage assay, proved to be crucial for explaining the assay difference. When platelet-rich plasma was used as the source of phospholipid in the one-stage assay, the factor VIII activity assay results correlated well with those measured by the chromogenic assay. Similar results were obtained when the platelets were replaced by liposomes prepared using platelet factor 3 (PF3) as a model that has a low content (5% to 10%) of phosphatidylserine (PS). In contrast, the use of liposomes with 20% to 30% PS, as in the crude lipid extracts used in ordinary APTT reagents, resulted in underestimation of the factor VIII activity. Antigen measurements using an enzyme-linked immunosorbent assay (ELISA) method demonstrated a good correlation between the antigen and chromogenic activity, but not always between antigen and one-stage activity results. Based on these findings and the clinical data, it can be concluded that the chromogenic assay most accurately measures the functional activity of BDDrFVIII. However, modifications of the one-stage assay, such as the use of a product-specific standard or development of a PF3-like phospholipid reagent, could address the observed assay discrepancies.