Protein palmitoylation represents the covalent linkage of a 16-carbon saturated fatty acid to a protein. This reversible post-translational modification directs protein-protein interactions as well as protein association with membranes and lipid rafts. Protein palmitoylation participates in ligand-induced signal transduction in several nucleated cells. Its role in platelet activation, however, has not previously been evaluated. We have found that platelets contain the palmitoyl transfer proteins GODZ and HIP14 as well as the palmitoyltransferase, acyl-protein thioesterase 1 (APT1). Thus, platelets possess the basic machinery for regulated palmitoylation. Studies using [3H]-labeled platelets identified several platelet proteins that were palmitoylated following exposure to the protease-activated receptor 1 (PAR-1) ligand, SFLLRN. To determine whether protein palmitoylation functions in activation-induced platelet functions, we infused recombinant APT1 into permeabilized platelets prior to activation with SFLLRN. Infusion of APT1 inhibited platelet protein palmitoylation and completely blocked platelet α-granule secretion induced by SFLLRN. Similarly, the protein palmitoylation inhibitor cerulenin blocked SFLLRN-induced platelet protein palmitoylation, α-granule secretion, and platelet aggregation in intact platelets. To assess the mechanism by which protein palmitoylation affects platelet function, we evaluated the effect of inhibitors of protein palmitoylation on G protein activity. Gαq is essential to PAR-1-mediated platelet activation and is palmitoylated in an activation-dependent manner in nucleated cells. Immunoprecipitation of Gαq from [3H]-labeled platelets showed that it is palmitoylated following activation of platelets with SFLLRN. Both APT1 and cerulenin inhibited SFLLRN-induced palmitoylation of Gαq. In addition, APT1 and cerulenin inhibited SFLLRN-induced GTPase activity as detected using [γ-32P]GTP-labeled platelet lysates. These results show that palmitoylation of Gαq participates in PAR-1-mediated signal transduction. We next used intravital microscopy to determine if protein palmitoylation functions in thrombus formation in vivo. For these experiments, platelets from a donor mouse were incubated with cerulenin and labeled with calcein-AM (green) or incubated with vehicle alone and labeled with calcein-AM red-orange (red). Equal numbers of green and red labeled platelets were then infused into a recipient mouse. The accumulation of cerulenin- and vehicle-treated platelets into thrombi following laser-induced injury of the mouse cremaster muscle was quantified using high speed, digital videomicroscopy. Incubation of platelets with cerulenin resulted in an approximately 50% reduction in their ability to accumulate into thrombi. These studies show that platelet protein palmitoylation is required for thrombus formation as well as for normal platelet function.