During blood-material interaction, the enzymes factor XII fragment (factor XIIf) and kallikrein are generated (contact activation). In this study, the enzymatic activities of factor XIIf and kallikrein were examined with an assay based on the conversion of tripeptide-p-nitroanilide substrate. With the use of aprotinin to inhibit kallikrein, the proteolytic activities of factor XIIf and kallikrein could be separately determined. In this in vitro study, two commercially available polyurethanes, Pellethane and Biomer®; three custom synthesized polyurethanes; a biomerlike 2000 Mw polytetramethyleneoxide containing polyurethane (PU-2000); an octadecyl extended (ODCE) biomer-like 2000 Mw Polytetramethyleneoxide containing polyurethane (PU-2000-ODCE); a hard-segment polyurethane (HS-PU); and glass (reference material) were incubated in 25% diluted plasma. In both series of experiments, glass caused the highest amidolytic activities by factor XIIf and kallikrein compared with any of the polyurethanes. In contrast, within the polyurethane group of materials, lower amidolytic activities by factor XIIf and kallikrein were measured on the custom-made polyurethanes than on the commercially available polyurethanes, although the differences among the polyurethanes were small. In addition, the influence of different ratios of material surface to the plasma incubation volume was studied. An increased ratio of surface area over plasma volume resulted in reduced contact activation, suggesting that plasma components are the limiting factor.