There are some controversies over the contribution of Na⁺/Ca²⁺ exchanger (NCX) to the regulation of cytosolic Ca²⁺ concentration ([Ca²⁺] _c) in smooth muscle. To prove the functional role of Na⁺/Ca²⁺ exchanger, we examined whether the removal of extracelluar Na⁺ could affect [Ca²⁺] _c of rabbit cerebral arterial smooth muscle. The fluorescence ratio of fura-2 (R_340/380) was measured in the single myocyte of rabbit middle cerebral artery and Na⁺ was substituted with the same concentration of NMDG⁺ or Li⁺. In 21 out of 230 cells tested, Na⁺-removal increased R_340/380 (ΔR_340/380) by 115±16.5% of the ΔR_340/380 induced by 10mM caffeine in the same cell. The Na⁺ removal-induced ΔR_340/380 was blocked by a selective inhibitor of cardiac type NCX exchanger (KB-R7943, (2- [2- [4-(4-nitrobenzyloxy) phenyl] ethyl] isothiourea, 10μM). In those cells where the Na⁺-removal by itself did not increase R_340/380, the caffeine-induced ΔR_340/380 was increased by Na⁺-removal (130±9.8% of control response, n=30). Under the whole-cell patch clamp condition, short application of caffeine induced transient increase of outward current (I_{k, Ca}-transient) which reflect the change of subsarcolemmal [Ca²⁺]. The application of KB-R7943 increased the amplitude of Ik, Ca-transient (n=4). These results suggest the functional existence of NCX in rabbit cerebral artery smooth muscle.