We have shown before that the lipid mediator sphingosine-1-phosphate (S1P) constricts isolated resistance arteries in a RhoA/Rho kinase-dependent manner and that myogenic constrictions are enhanced after overexpression of sphingosine kinase (Bolz et al. 2003), the enzyme generating S1P. Moreover, it has been reported that myogenic vasoconstriction requires the generation of reactive oxygen species (ROS) (Novicki et al. 2001). Here we studied, whether S1p is able to induce ROS production and whether this contributes to myogenic constriction. All experiments were performed in resistance arteries (diameter 100-200 μm) isolated from hamsters killed by an overdose of pentobarbital. Transfection of the arteries was performed using the transfectant effectene (60 μl/3ml + 5 μg DNA) over 20 h in culture before the start of the experiments. The cannulated arteries were perfused, their smooth muscle loaded with the calcium indicator Fura2-AM (2 μM) or the ROS indicator dichloro-fluorescein (10 μM) as described before (Bolz et al. 2003). Myogenic constrictions were induced by an increase of hydrostatic pressure from 45 to 110 mm Hg. Values are given as means ± SEM, significance was tested using the t test for paired or unpaired data. P values <0.05 were considered significant. Elevation of the transmural pressure induced a myogenic constriction (106 ±6% reversal of the pressure induced distension (RID), n=6 ) as well as ROS production of resistance artery smooth muscle by 42±10% (as observed 2 mins after pressure increase, n=4). Pressure-induced increase in ROS production was significantly higher (134±29%) after overexpression of Sphk1 (n=4) and virtually abolished after expression of its dominant negative mutant, hSK G82D (n=4) . In the latter vessels exogenous S1P (1μM) still was able to stimulate ROS generation siginificantly (by 2.8±0.3-fold, n=6). The initial (2 min) pressure-induced ROS elevation was completely inhibited by the NADPH oxidase inhibitor dephenyleneiodonium (1μM), which also abolished myogenic vasoconstriction (-14±7% of RID). Transfection (n=5) of dominant negative Rac mutant (N17Rac), also prevented the initial pressure-induced ROS formation and abolished myogenic vasoconstriction. These results point towards a mandatory role for Sphk1/S1P in mediating pressure-induced ROS formation which involves activation of Rac. The S1P dependent, pressure-induced ROS formation plays a pivotal role in the regulation of myogenic reactivity of microvessels.