Cytoplasmic free Ca²⁺ ([Ca²⁺]_cyt) is essential for the contraction and relaxation of blood vessels. The role of plasma membrane Na+/Ca²⁺ exchange activity in the regulation of vascular Ca²⁺ homeostasis was previously ascribed to the NCX1 protein. However, recent studies suggest that a relatively new K⁺-dependent Na⁺/Ca²⁺ exchanger, NCKX (gene family SLC24), is also present in vascular smooth muscle. The purpose of present study was to identify the expression and function of NCKX in arteries. mRNA encoding NCKX3 and NCKX4 was demonstrated by RT-PCR and Northern blot in both rat mesenteric and aortic smooth muscle. NCXK3 and NCKX4 proteins were also demonstrated by immunoblot and immunofluorescence. After voltage-gated Ca²⁺ channels, store-operated Ca²⁺ channels and Na⁺ pump were pharmacologically blocked, when the extracellular Na⁺ was replaced with Li⁺ (0 Na⁺) to induce reverse mode (Ca²⁺ entry) activity of Na⁺/Ca²⁺ exchangers, a large increase in [Ca²⁺]cyt signal was observed in primary cultured aortic smooth muscle cells. About half of this [Ca²⁺]cyt signal depended upon the extracellular K+. In addition, after the activity of NCX was inhibited by KB-R7943, Na⁺ replacement-induced Ca²⁺ entry was absolutely dependent on extracellular K⁺. In arterial rings denuded of endothelium, a significant fraction of the phenylephrine-induced and nifedipine-resistant aortic or mesenteric contraction could be prevented by removal of extracellular K⁺. Taken together, these data provide strong evidence for the expression of NCKX proteins in the vascular smooth muscle, and their novel role in mediating agonist-stimulated [Ca²⁺]_cyt and thereby vascular tone.