The purpose of this study was to examine the effect of the in vivo maturing ePTFE graft surface on platelet activation. Ten canines were randomized to receive either a carotid to infrarenal aorta ePTFE graft or sham operation. Animals were sampled at specific time points up to 3 months postoperatively. Whole blood platelet aggregometry (arachidonic acid, ADP, and collagen agonists) and ATP secretion (in response to arachidonic acid, ADP, collagen, and thrombin) were measured. Additionally, complete hematologic analysis and histology were performed. With time, graft animals showed significantly more decrease in platelet aggregation in response to ADP compared to sham animals (P = .023). The total amount of ATP per platelet was not different, as demonstrated by equivalent ATP release per platelet in response to thrombin. Over the first week, grafted dogs developed a decrease in systemic platelet count of 50% (P < .001) that persisted over the 3-month follow-up period. With time, overall regression model slopes of graft and sham platelet count data were not statistically different (P = .29). Histologically, the grafts demonstrated limited cellular ingrowth at both anastomoses, with fibrin matrix along the remainder of the blood-biomaterial interface. These data suggest that, similar to Dacron, exposure to an ePTFE surface results in significant changes in platelet biology, and these platelet-ePTFE interactions persist even after the graft has formed a mature pseudointima. The pseudointima appears to be the primary determinant of the blood-biomaterial interaction.