The pro-angiogenic effects of vascular endothelial growth factor (VEGF) are key to maintaining normal microvasculature and preventing injury. VEGF is present in the platelet proteome and released from thrombin-stimulated platelets in vitro, indicating that platelets may be significant physiological transporters of VEGF. We studied this possible function by examining the ability of platelets to release VEGF under a range of conditions, and by establishing the intracellular location of VEGF relative to other platelet proteins. Western blot analysis was used to measure platelet release of VEGF in vitro in response to stimulation with agonists both weak (adenosine diphosphate, epinephrine) and strong (thromboxane A2 analog U46619, PAR-1 thrombin receptor activating peptide). Both classes of agonist stimulated VEGF release, indicating that strong stimuli (e.g. thrombin) are not necessarily required for platelet VEGF release in vivo. The intracellular location of platelet VEGF and its colocalization with other platelet proteins was determined using immunohistochemical techniques and spinning-disc confocal laser fluorescence microscopy, combined with computerized image processing and colocalization analysis. Platelet VEGF was observed to be distributed throughout the platelet interior in small, numerous (>30/cell) concentrations resembling ?-granules. However, VEGF did not show colocalization with either of the known ?-granule proteins von Willebrand factor or fibrinogen, nor with several other dense granule, organelle, receptor and cytoskeletal proteins. Thus platelet VEGF appears to represent a unique protein pool that is released in response to a wide range of platelet stimuli.