Fura-2 fluorescence measurements of cytosolic free Ca²⁺ concentration have led to the hypothesis that depletion of intracellular Ca²⁺ stores in the A7r5 rat aortic smooth muscle cell line activates a Ca²⁺ entry mechanism known as capacitative or store-operated Ca²⁺ entry (CCE or SOCE, respectively). In a recent study (1), we described a store-operated current (I_SOC) in A7r5 cells, which was activated by passive depletion of Ca²⁺ stores with 1 μM thapsigargin or by active store depletion with 1-100 nM AVP. Because of different recording conditions, it has not been previously determined whether I_SOC corresponds to CCE measured using fura-2. Nor has the channel protein(s) responsible for this Ca²⁺ entry pathway been identified. In the present study, the pharmacological characteristics of I_SOC, including its inhibition by 2-aminoethoxydiphenylborane (2-APB), diethylstilbestrol, or micromolar Gd³⁺, were found to parallel the effects of these drugs on CCE measured under identical external ionic conditions using fura-2 (Fig. 1). Rat mesenteric artery smooth muscle cells (MASMC) were prepared from arteries isolated from Sprague-Dawley rats anaesthetized with isolflurane (4% by inhalation); rats were then humanely killed. Thapsigargin-stimulated I_SOC in freshly isolated MASMC was similar to that recorded from A7r5 cells. Members of the TRP family of non-selective cation channels, TRPC1, TRPC4, and TRPC6 were detected by RT-PCR and Western blot in both A7r5 cells and MASMC. A stable A7r5 cell line expressing a small interfering RNA, which selectively reduced TRPC1 mRNA expression (by 43 ± 7% (mean ± S.E.M.), determined using real-time RT-PCR), exhibited significantly decreased thapsigargin-stimulated I_SOC (inward current measured at -100 mV (mean ± standard error): -0.21 ± 0.02 pA/pF (n=7), compared with -0.49 ± 0.05 pA/pF (n=8) measured in control cells; p<0.001, Student's t test), suggesting that this channel contributes to the store-operated pathway.