Clarity about the nitric oxide (NO) concentrations existing physiologically is essential for developing a quantitative understanding of NO signalling, for performing experiments with NO that emulate reality, and for knowing whether or not NO concentrations become abnormal in disease states. A decade ago, a value of about 1 microM seemed reasonable based on early electrode measurements and a provisional estimate of the potency of NO for its guanylyl cyclase-coupled receptors, which mediate physiological NO signal transduction. Since then, numerous efforts to measure NO concentrations directly using electrodes in cells and tissues have yielded an irreconcilably large spread of values. In compensation, data from several alternative approaches have now converged to provide a more coherent picture. These approaches include the quantitative analysis of NO-activated guanylyl cyclase, computer modelling based on the type, activity and amount of NO synthase enzyme contained in cells, the use of novel biosensors to monitor NO release from single endothelial cells and neurones, and the use of guanylyl cyclase as an endogenous NO biosensor in tissue subjected to a variety of challenges. All these independent lines of evidence suggest the physiological NO concentration range to be 100 pM (or below) up to approximately 5 nM, orders of magnitude lower than was once thought.