Almost a quarter of a century ago Casteels & Droogmans (1981) published their seminal work on arterial smooth muscle showing that depletion of intracellular stores by noradrenaline stimulated the rate of calcium uptake. The pathway was resistant to conventional calcium antagonists and inhibited by potassium-induced depolarisation. Subsequently many studies have shown this is a common phenomenon in smooth muscle but the underlying mechanisms and functional importance remain uncertain. Evidence has however accumulated that TRPC1 protein, a mammalian homologue of Drosophila transient receptor potential, is a component of the channel (Beech et al 2003). We have studied the phenomenon in rabbit pial arterioles and support a role for TRPC1 (Xu & Beech 2001). Rabbits were killed humanely according to approved schedule 1 procedures. Intriguingly, however, TRPC1 forms heteromultimeric channel assemblies with TRPC5 (Strubing et al 2001), which is potentially important because there is TRPC5-encoding mRNA in pial arterioles (Flemming et al 2003). Therefore, we tested the hypothesis that TRPC1 operates in concert with TRPC5 in the native store-operated calcium-entry mechanism of arterioles. In order to test for TRPC5 protein, three new anti-TRPC5 antibodies were prepared. All antibodies labelled over-expressed TRPC5 and specifically bound a protein of the same mass in blood vessels. There was evidence of glycosylated TRPC5 and plasma membrane localisation. One of the antibodies was designed to a unique amino acid sequence in the predicted outer vestibule of the ion pore of TRPC5. Strikingly, the antibody ablated calcium-entry through TRPC5 over-expressed in HEK-293 cells and calcium-entry evoked by store depletion in arterioles, without effect on background calcium entry. The antibody also suppressed a stimulatory effect of lanthanum in store-depleted arterioles, consistent with the unusual response of TRPC5 to this lanthanide. We demonstrate the antibody is highly specific for TRPC5, being able to distinguish it from the closely related proteins TRPC1, TRPC4 and TRPC6. From these data it is suggested that TRPC5 protein is expressed in vascular smooth muscle and that we have engineered a novel tool for the study of its function. Most significantly we demonstrate TRPC5 is an additional and critical subunit of the physiological store-operated calcium-entry mechanism in vascular smooth muscle.