Calcium entry mechanisms have important roles in vascular smooth muscle cells, regulating contraction, phenotype, proliferation and motility. Recent studies suggest two types of relatively unexplored membrane protein, STIM1 and Orai1, function together in calcium release-activated calcium (CRAC) channels of lymphocytes and cell lines (Liou et al. 2005; Feske et al. 2006; Spassova et al. 2006; Mercer et al.2006). Here we explore the expression and function of STIM1, Orai1 and their homologues in vascular smooth muscle cells from discarded saphenous vein samples obtained with ethical approval during coronary artery bypass surgery. Messenger RNA species encoding STIM1, STIM2, Orai1, Orai2 and Orai3 were detected in order of abundance: Orai3>Orai1>Orai2>STIM1>STIM2. siRNA probes were screened for efficacy against each RNA species and probes causing >75% specific reduction of RNA abundance were used for further experiments. Calcium measurements were made on a real-time fluorescence 96-well plate reader. Knock-down of RNA encoding STIM1 or STIM2 suppressed calcium entry after store depletion. Ca2+-content of intracellular stores, as determined by agonist-evoked Ca2+-release, was unaffected by STIM1 siRNA. Antibody targeted to STIM1 also inhibited calcium entry. Anti-STIM1 staining was localized to small spots that were reduced by siRNA targeting STIM1. The number of spots did not change significantly in response to store-depletion. Knock-down of RNA encoding Orai1 also suppressed calcium entry but no effects relating to RNA encoding Orai2 or Orai3 were detected. The data suggest two new families of membrane protein with functional importance for calcium entry in vascular smooth muscle.