Previous studies have shown that anticoagulants alter platelet reactivity and the apparent effects of antiplatelet agents. This study was conducted to identify the impact of methods of preparation of blood samples on an assay of platelet function and the effects of antiplatelet agents. The activation of platelets was identified with the use of flow cytometry in response to thrombin (1 and 10 nmol/L), adenosine diphosphate (0.2 and 1 μmol/L), platelet activating factor (1 nmol/L), and convulxin (1 and 10 ng/ml). Antiplatelet effects were assessed after the addition in vitro of tirofiban (50 ng/ml) and cangrelor (10 nmol/L). Results were compared in whole blood and platelet-rich plasma (PRP) anticoagulated with corn trypsin inhibitor (32 μg/ml, a specific inhibitor of factor XIIa). The fraction of young platelets was quantified with thiazole orange, which identifies ribonucleic acid. The activation of platelets was consistently less in PRP compared with whole blood. Activation in PRP was 23 ± 15% that in whole blood for thrombin, 65 ± 26% for adenosine diphosphate, 40 ± 20% for platelet activating factor, and 49 ± 25% for convulxin (p <0.01 for each comparison). The fraction of young platelets in PRP was 39 ± 11% that in whole blood (p <0.001). The effects of antiplatelet agents varied with agonist and antiplatelet agent but were generally greater in PRP compared with whole blood (p <0.05). In conclusion, platelet reactivity is lower and the effects of antiplatelet agents are greater and potentially misleading in PRP compared with whole blood. The accuracy of platelet function testing may be improved by performance in whole blood.