The platelet-specific chemokine platelet factor 4 (PF4) is released in large amounts at sites of vascular injury. PF4 binds to heparin with high affinity, but its in vivo biologic role has not been defined. We studied the role of PF4 in thrombosis using heterozygote and homozygote PF4 knock-out mice (mPF4^+/– and mPF4^–/–, respectively) and transgenic mice overexpressing human PF4 (hPF4⁺). None of these lines had an overt bleeding diathesis, but in a FeCl₃ carotid artery thrombosis model, all showed impaired thrombus formation. This defect in thrombus formation in the mPF4^–/– animals was corrected by infusing hPF4 over a narrow concentration range. The thrombotic defect in the mPF4^+/– and mPF4^–/– animals was particularly sensitive to infusions of the negatively charged anticoagulant heparin. However, the same amount of heparin paradoxically normalized thrombus formation in the hPF4⁺ animals, although these animals were anticoagulated systemically. Upon infusion of the positively charged protein, protamine sulfate, the reverse was observed with mPF4^+/– and mPF4^–/– animals having improved thrombosis, with the hPF4⁺ animals having worsened thrombus formation. These studies support an important role for PF4 in thrombosis, and show that neutralization of PF4 is an important component of heparin's anticoagulant effect. The mechanisms underlying these observations of PF4 biology and their clinical implications remain to be determined.