Nitric oxide (NO) is a potent vasodilating agent implicated in cochlear blood flow regulation. We recently demonstrated that exogenously applied NO donor DPTA-NONOate hyperpolarizes both endothelial and smooth muscle cells of in vitro spiral modiolar artery (SMA) via activation of ATP-sensitive K+ channels (K(ATP)). Also, NO was detected in the SMA cells by NO indicator dye in the in vitro basal condition. Using intracellular recording techniques, electrochemical NO-sensing measurement, and a vaso-diameter video tracking method, we investigated the basal release of NO from the in vitro SMA and its role in the vascular function. We found that (1) 300 microM L-NAME, a NO synthase inhibitor, and 3 microM glipizide caused a depolarization of approximately 4.5 and approximately 3.2 mV, respectively, in cells with a resting potential less negative than -60 mV; (2) NO sensor in the close vicinity of the SMA detected a NO concentration of approximately 50 nM that was suppressed by L-NAME and enhanced by L-arginine (1-1000 microM); (3) NO donor DPTA-NONOate (0.1-30 microM) applications produced about 8-245 nM of NO in the recording bath. These data indicate a NO concentration-hyperpolarization relation, with an EC50 of 22 nM. (4) Finally, L-NAME but not glipizide produced a 4.8% reduction in SMA diameter (approximately 50 microm) in the majority of SMAs, whereas NONOate (10 microM) always caused a dilation. Both the induced constriction and dilation were not significantly affected by 3 microM glipizide. We conclude that a significant amount of NO (> 50 nM) is tonically released from the in vitro SMA, which is above the EC50 for activation of K(ATP), and thus contributes to the membrane polarization. The basal release of NO also contributes to vasotone relaxation, but the K(ATP) activation appears to play little role in the relaxation of the in vitro SMA.