Summary. When the one-stage clotting assay is used in comparison with the chromogenic and immunological assays, plasma levels of factor (F)VIII are underestimated by 40–50% after infusion of B-domain deleted recombinant FVIII (BDD-rFVIII) in patients with hemophilia. A possible way to counteract the underestimation of FVIII levels by the one-stage assay is the adoption of a recombinant FVIII reference standard instead of a plasma standard. To evaluate the usefulness of such a standard [ReFacto® Laboratory Standard (RLS)], the pharmacokinetic parameters of a single dose of BDD-rFVIII (25 U kg−1) were evaluated in a multicenter study carried out in 18 patients with severe hemophilia A. The very low in vivo recovery, obtained with the combination of the one-stage assay and plasma reference standard, was increased up to the values obtained by the chromogenic assay when the results were expressed in terms of RLS. When the plasma standard was used, the one-stage/chromogenic ratio was 0.82 ± 0.12 for FVIII levels above 25 U dL−1 and 1.42 ± 0.99 for FVIII levels below 25 U dL−1. Using the RLS, the one-stage/chromogenic ratio increased to 1.01 ± 0.19 at FVIII levels above 25 U dL−1, as a consequence of a complete overlap of the two decays; however, at FVIII levels below 25 U dL−1, the one-stage/chromogenic ratio was still 1.6 ± 0.85. After the twelfth hour, FVIII concentrations obtained by chromogenic assay were always lower than those resulting from the one-stage clotting assay, independently of the standard used. Results obtained by chromogenic assay were not affected by the type of standard used. Compared with those obtained by the one-stage assay, higher values of clearance, lower volume of distribution area and shorter plasma half-life or mean residence time were obtained by chromogenic assay because of a shape change of the decay curve due to a shift to higher values in the first part (time interval 0–12 h) and to lower values in the second part of the decay curve (time interval 12–48 h). As a consequence, the slope of the decay curve obtained by means of chromogenic assay was steeper. In conclusion, the more homogeneous results of in vivo recovery and pharmacokinetic analysis, due to the decrease of discrepancy between the two methods when RLS was used, make the cheaper and more widely used one-stage assay preferable to the more expensive chromogenic assay, on condition that the ReFacto specific standard has used.