Methods for evaluating bovine platelet activation, such as the 14C-serotonin release assay, flow cytometric determination of membrane protein expression, and quantification of protease activated receptor-1 (PAR-1) cleavage, have been developed to enable the investigation of the activation and signaling mechanisms in bovine platelets. The concurrent monitoring of membrane expression, granular release, phosphorylation reactions, and aggregation in platelet suspensions, prepared from the same donors, stimulated with agonists such as [alpha]-thrombin, and [gamma]-thrombin has facilitated comparative studies on the signaling and activation mechanisms in bovine and human platelets. Incubation of sub-optimal concentrations of [alpha]-thrombin, [gamma]-thrombin, and platelet activation factor with platelets pre-treated with signaling enzyme inhibitors: staurosporine; okadaic acid; wortmannin; and genistein, was utilized in determining the roles of protein kinases, serine threonine phosphatases, phosphatidylinositol-3-kinase, and tyrosine kinases respectively, in the propagation of bovine and human platelet activation. These studies reveal that bovine and human platelets possess the same major signaling molecules but exhibit differential responses to the same agonists. Both bovine and human platelet activation may be mediated by protein kinase dependent processes. Phosphatidyinositol-3-kinase contributes but does not play a primary role in the activation process in either platelet species. In contrast, serine threonine phosphatases are critical for only human platelet activation and not for bovine platelet activation. The lack of response of bovine platelets to PAR-1 activating peptides, SFLLRN and TR1-41, and their increased sensitivity to [gamma]-thrombin suggests that the primary thrombin receptor on bovine platelets may not be PAR-1. Unlike bovine platelets, both [alpha]- and [gamma]-thrombin-mediated activation in human platelets are mediated by the same signaling pathways suggesting differences in the signaling mechanisms between both species. In bovine platelets discrete signaling pathways influence each of the activation markers whereas in human platelets, all the markers of activation are influenced by the same signaling molecules and/or pathways. Comparative investigations on signaling mechanisms in bovine and human platelets provide a platform for further research in the roles of mammalian platelets in thrombosis and possibly inflammation.