Abstract--The role of NO in the regulation of the mechanical properties of conduit arteries is controversial in humans, and the involvement of an endothelium-derived hyperpolarizing factor (EDHF), acting through calcium-activated potassium (K_Ca) channels, has never been investigated at this level in vivo. We assessed in healthy volunteers, after oral administration of aspirin (500 mg), the effect of local infusion of N^G-monomethyl-L-arginine (L-NMMA; 8 µmol/min for 8 minutes), an NO synthase inhibitor, tetraethylammonium chloride (TEA; 9 µmol/min for 8 minutes), a K_Ca channels inhibitor, and the combination of both on radial artery internal diameter, wall thickness (echo tracking), blood flow (Doppler), and pressure. The incremental elastic modulus and compliance were fitted as functions of midwall stress. L-NMMA decreased modulus and increased compliance at high levels of midwall stress (all P<0.05) without affecting radial diameter. TEA reduced radial diameter from 2.68±0.07 to 2.50±0.08 10^-3 m, increased the modulus, and decreased the compliance at all levels of stress (all P<0.05). Combination of both inhibitors synergistically enhanced the increase in modulus, the decrease in diameter (from 2.71±0.10 to 2.42±0.09 10^-3 m), and compliance compared with TEA alone (all P<0.05). These results confirm that inhibition of NO synthesis is associated with a paradoxical isometric smooth muscle relaxation of the radial artery. They demonstrate the involvement of K_Ca channels in the regulation of the mechanical properties of peripheral conduit arteries, supporting a role for EDHF at this level in vivo. Moreover, the synergistic effect of L-NMMA and TEA shows that K_Ca channels compensate for the loss of NO synthesis to maintain peripheral conduit artery diameter and mechanics.