Anticoagulants used for platelet function studies in vitro may affect platelet responsiveness. In the present study we compared the influence of three different anticoagulants, sodium citrate, low molecular mass heparin, and recombinant hirudin, on platelet aggregation in whole blood in vitro, using impedance aggregometry. ADP and collagen induced aggregation was significantly lower in citrated blood compared to hirudin treated blood, reflecting the importance of extracellular calcium for platelet function. Inhibition of platelet aggregation by aspirin, was more pronounced in citrated blood compared to hirudin treated blood, in agreement with the concept of artifactually enhanced thromboxane generation in media containing low extracellular calcium levels. In blood anticoagulated with low molecular mass heparin, platelet aggregation to collagen tended to be enhanced as compared to hirudin treated blood, whereas platelet responses to ADP at a high concentration were slightly reduced. Of the anticoagulants investigated, the selective thrombin inhibitor hirudin is the most suitable anticoagulant for studies of platelet aggregation in vitro in whole blood.