Tyrosine phosphorylation of the non-receptor tyrosine kinases pp72syk and pp125FAK and of the gamma2 isoform of phospholipase C (PLCgamma2) in human platelets stimulated with the lectin Concanavalin A was investigated. Concanavalin A induced the rapid tyrosine phosphorylation of pp72syk and PLCgamma2 with a similar kinetics, while tyrosine phosphorylation of pp125FAK occurred in a later phase of platelet activation. When compared with other platelet agonists, Concanavalin A revealed to be at least as potent as collagen in inducing tyrosine phosphorylation of PLCgamma2 and pp125FAK, while tyrosine phosphorylation of pp72syk induced by the lectin was much stronger than that induced by thrombin or collagen. Concanavalin A-induced tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK was not dependent on platelet aggregation as it occurred normally even in the absence of sample stirring and when fibrinogen binding to integrin alphaIIb-beta3 was inhibited by the peptide RGDS. Tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK required the binding of the lectin to the platelet surface, but was not observed in platelets treated with succinyl-Concanavalin A, a derivative of the lectin that interacts with the same receptors but does not promote clustering of membrane glycoproteins. Moreover, the aggregation-independent tyrosine phosphorylation of pp125FAK and pp72syk induced by Concanavalin A required the expression of integrin alphaIIb-beta3 on the platelet surface as it was strongly inhibited in platelets from patients affected by Glanzmann thrombasthenia. By contrast, tyrosine phosphorylation of PLCalpha2 occurred normally also in thrombasthenic platelets stimulated with Concanavalin A. These results demonstrate that, even in the absence of aggregation, the clustering of integrin alphaIIb-beta3 induced by Concanavalin A on the platelet surface directly promotes tyrosine phosphorylation of pp72syk and pp125FAK and provide further evidence that the oligomerization of the fibrinogen receptor promoted by its natural ligand during platelet aggregation may be responsible for the tyrosine phosphorylation of these proteins induced by physiological agonists.