The continuous release of the endothelial autacoid nitric oxide is known to inhibit platelet adhesion and aggregation. The endothelium releases also one or more hyperpolarising factors (EDHF), which in certain vessels were characterised as epoxyeicosatrienoic acid(s) (EET) being produced by cytochrome (CYP) P450 (1). We have shown before that a CYP2C9 product mediates EDHF induced vasodilation in isolated hamster resistance vessels (2). We investigated whether EETs/EDHF act as inhibitors of platelet adhesion in vitro and in vivo. Experiments were performed using washed human platelets freshly obtained from healthy donors. Platelet adhesion was investigated on monolayers of human endothelial cells (HUVEC) and by intravital microscopy in arterioles of awake hamsters (dorsal skinfold chamber preparation, injection of fuorescence-labeled human platelets). Values are presented as means ±SEM. The resting platelet membrane potential (using the fluorescent dye DiBac4) amounted to -58 ±9mV (FACS analysis, n=30). 11,12-EET (1μM) significantly (t-test) hyperpolarized platelets to -69± 2 mV (n=12), which was prevented by the K⁺ channel inhibitors charybdotoxin (50nM, n=4) and iberiotoxin (500nM, n=4) whereas apamin had no effect. 11,12 EETs also inhibited platelet adhesion to HUVEC under static and flow conditions by more than 20% (n=13-20). Stable overexpression of cytochrome CYP2C9 in EA.hy926 cells (a commercially available, immortalised cell line with some HUVEC properties) resulted in release of a factor that hyperpolarized platelets and inhibited their adhesion. Exposure to EETs inhibited platelet P-selectin expression in response to ADP. In vivo, the velocity distribution of circulating platelets (measured in 4-6 arterioles each of 6 animals) was significantly shifted to the left (lower velocities) after superfusion with a specific inhibitor of CYP2C9 (sulfaphenazole 100 μM). EETs hyperpolarize platelets and inhibit their adhesion to the endothelium in a membrane potential-dependent manner. They are released from CYP2C9 expressing endothelial cells and act as an EDHF on platelets in vitro. In the hamster microcirculation, inhibition of CYP2C9 induces a significant reduction of platelet velocities indicating that EDHF may also play an antiadhesive role in vivo.