Introduction: Platelet activation/aggregation plays a key role in the dysregulation of coagulation and the development of thrombotic microangiopathy in nonhuman primate recipients of pig xenografts. As a preliminary to the study of anti-platelet therapy in vitro and in vivo, the present study aimed to compare platelet aggregation in whole blood from humans, baboons, and cynomolgus monkeys. Methods: Using "Chrono-log" technology (two-sample four-channel Chrono-log Whole Blood Aggregometer), we studied aggregation of platelets in healthy humans (n = 8), baboons (n = 5), and monkeys (n = 8). Whole blood (WB) samples were collected, and platelet aggregation was assessed using three different volumes of blood (1, 0.5, and 0.25 ml). Platelet activation was induced using collagen (at 3 and 5 μg/ml), ristocetin (at 0.5 and 1.0 mg/ml), adenosine diphosphate (ADP; at 10, 20, and 40 μm), or thrombin (at 1 and 5 IU/ml). Inhibition of agonist-induced platelet aggregation by heparin and low molecular weight heparin (LMWH) (at 1, 10, and 100 IU/ml) was evaluated. Results: Mean platelet counts were 222.1, 263.2, and 276.1 (× 10 3/μl) in humans, baboons, and monkeys, respectively. In all three species, platelet aggregation was induced by collagen, ristocetin, ADP, or thrombin in a dose-dependent manner. A blood volume of 0.5 ml provided the most consistent results with all agonists in all three species. Dilution studies indicated that there was a significant positive correlation between platelet count and percent aggregation of platelets (P < 0.05). Collagen (3 and 5 μg/ml), ADP (10, 20, and 40 μm), and thrombin (1 and 5 IU/ml) induced significantly greater platelet aggregation in humans than in baboons. ADP (20 and 40 μm) and thrombin (1 and 5 IU/ml) induced significantly greater platelet aggregation in monkeys than in baboons. There was no species difference with ristocetin (0.5 or 1.0 mg/ml). In all species, thrombin (1 or 5 IU) induced greater platelet aggregation than any of the other reagents. Heparin at 1 IU/ml and LMWH at 10 IU/ml in all species almost completely abrogated thrombin-induced platelet aggregation. Heparin at 100 IU/ml effectively inhibited platelet aggregation induced by collagen, but only partially inhibited aggregation induced by ADP or ristocetin. LMWH only partially inhibited aggregation induced by collagen, ristocetin, and ADP. Conclusions: The "Chrono-log" technology proved to be a reliable method of evaluating platelet activation and aggregation in vitro in primates. Species differences may play a role in platelet aggregation, with the monkey being more comparable to the human than the baboon, although overall trends were similar. In all species, thrombin induced greater platelet aggregation than other agonists. Even a concentration of heparin of 1 IU/ml, which is probably the maximal concentration that is clinically-applicable, prevented platelet aggregation induced by thrombin, but was less effective in preventing aggregation induced by collagen, ADP, or, particularly, ristocetin. © 2012 John Wiley & Sons A/S.