Two α-isoforms of the Na+-K+-ATPase are expressed in vascular smooth muscle cells (VSMCs). The α1-isoform is proposed to serve a cytosolic housekeeping role, whereas the α2-isoform modulates Ca2+storage via coupling to the Na+-Ca2+exchanger (NCX) in a subsarcolemmal compartment. To evaluate the ramifications of this proposed interaction, Ca2+-store load and the contributions of the primary Ca2+transporters to Ca2+clearance were studied in aortic VSMCs from embryonic wild-type (WT) and Na+-K+-ATPase α2-isoform gene-ablated, homozygous null knockout (α2-KO) mice. Ca2+stores were unloaded by inhibiting the sarco(endo)plasmic reticulum Ca2+-ATPase with cyclopiazonic acid (CPA) in Ca2+-free media to limit Ca2+influx. Ca2+clearance by the plasma membrane Ca2+-ATPase (PMCA), NCX, or mitochondria was selectively inhibited. In WT VSMCs, NCX accounted for 90% of the Ca2+efflux. In α2-KO VSMCs, preferential clearance of store-released Ca2+by NCX was lost, whereas PMCA activity was increased. Selective inhibition of the α2-isoform (0.5 μM ouabain for 20 min), before treatment with CPA enhanced the store load in VSMCs from WT, but not α2-KO mice. A subsequent analysis of capacitative Ca2+entry (CCE) indicated that the magnitude of Ca2+influx was significantly greater in α2-KO cells. Our findings support the concept of a subsarcolemmal space where the α2-isoform coupled with NCX modulates Ca2+-store function and, thereby, CCE.