Hydrogen-peroxide (H2O2) has been reported to either relax or constrict blood vessels depending on species and vessel type. Arteriolar responses (internal diameter (i.d.) and changes in [Ca2+]i by Fura-2 fluorescence ratio (F340/380)) were determined in isolated, cannulated and pressurized (80 mmHg) arterioles obtained from rat skeletal muscle (i.d.: 168±6 µm, n=50), from rat coronaries (i.d.: 120±9 µm, n=6), or from human coronaries (i.d.: 138±29 µm, n=5). Wistar rats (male, 250-350 g, n=50) were anaesthetized with an I.P. injection of pentobarbital-natrium (150 mg/kg). Human arterioles were dissected from right atrial appendages of patients (n=5) who underwent cardiac surgery. Data are shown as average diameter ± S.E.M., compared by Student’s t-test or ANOVA. Arteriolar constrictions were expressed as changes in i.d. as a percentage of the baseline diameter, dilations were represented as changes in i.d. as percentage of maximal dilation. H2O2 evoked biphasic responses in skeletal muscle arterioles: constriction at lower (10 µM-100 µM, 34±3% at 100 µM, p<0.001) and dilation at higher concentrations (1 mM-10 mM, 80±11% at 10 mM, p<0.001). In contrast, H2O2 (10 mM) caused only vasodilation in rat (96±3%, p<0.001) and human (94±14%, p=0.01) coronary arterioles. The H2O2 evoked constriction in skeletal muscle arterioles was not accompanied by significant changes in F340/380 in a range of H2O2 concentrations between 1 µM and 100 µM, but higher concentrations of H2O2 elicited significant decreases in [Ca2+]i (decrease in F340/380 from 1.13±0.03 to 1.01±0.01 at 1 mM H2O2, p<0.05). In skeletal muscle arterioles 100 µM H2O2 induced constrictions were inhibited in the absence of endothelium (0±8% constriction, p=0.03). Moreover, in the presence of antagonists of: thromboxane A2 (1 µM SQ-29548), cyclooxygenase (10 µM indomethacin), or protein tyrosine phosphatase (5 µM α-Bromo-4-methoxyacetophenone) the 100 µM H2O2-induced constrictions were converted to dilations (44±11%; 50±17%, or 22±17%, respectively, p<0.005). Furthermore, antagonists of phospholypase A (100 µM 7,7-Dimethyl-(5Z,8Z)-eicosadienoic acid), protein kinase C (10 µM chelerytrine), phospholypase C (1 µM U-73122), or src kinase (1 µM Src inhibitor-1) decreased the 100 µM H2O2-induced constriction (9±2%, 9±4%, 4±2%, 8±3%, respectively, p<0.005). Our data suggest that in skeletal muscle arterioles H2O2 increases the Ca2+ sensitivity of the contractile apparatus of arteriolar smooth muscle and activate the endothelial src kinase, phospholypase C, protein kinase C, phospholypase A, cyclooxygenase pathway leading to the synthesis of thromboxane A2 an hence generate constriction. In contrast, in coronary arterioles this pathway is not functional, suggesting differences in H2O2 mediated physiological responses in particular endothelium-derived hyperpolarizing factor like effects in these vessels.