Controversy exists as to whether a fall in the intracellular Ca2+ concentration ([Ca2+]i) is a requisite element of the vasodilatory response to nitric oxide (NO). We studied the effect of NO on the coupling between [Ca2+]i and vasoconstriction in arterial segments loaded with the [Ca2+]i‐sensitive, intracellular dye fura‐2. As data interpretation is equivocal when fura‐2 is loaded into both endothelial and smooth muscle cells, we compared results from in vitro experiments on segments of the rat tail artery in which fura‐2 and noradrenaline were applied on the luminal or adventitial side, and endothelium was removed ‘physically’ (rubbing or air) or ‘functionally’ (Nω‐nitro‐l‐arginine methyl ester). The use of air perfusion to remove endothelium is of considerable benefit since it allows paired observations in a single tissue. Fura‐2 loaded into endothelial cells but endothelial ‘contamination’ of the smooth muscle cell [Ca2+]i signal was minimal. Endogenous NO decreased vasoconstrictor responses to noradrenaline but had no effect on [Ca2+]i. Nitroglycerine decreased vasoconstrictor responses in a concentration‐dependent fashion but had no effect on [Ca2+]i. In conclusion, NO causes vasodilatation via a mechanism which is downstream of [Ca2+]i mobilization.