Key points The metabolic theory of blood flow regulation suggests that, when tissue cells experience a reduction of oxygen supply, they produce metabolic vasodilators, which increase the lumen of arterioles and hence local blood flow. A century of intensive research has not found a single metabolic vasodilator to account for observed flow changes, therefore current thought is that there are many vasodilators. We have proposed an alternative hypothesis based on the interaction of two well‐known molecular mechanisms for generation and removal of the intercellular signalling radicals nitric oxide (NO) and superoxide. The proposed mechanism of regulation predicts a sharp increase in NO concentration in the intercellular space at the onset of muscle contraction. Experiments with the NO‐sensitive fluorescent indicator DAF‐FM, loaded into the intercellular space, confirmed the rapid response of the NO‐related signal at the beginning of contractions and rapid washout of the indicator after their termination. Abstract The prevailing metabolic theory of local blood flow regulation suggests the dilatation of arterioles in response to tissue hypoxia via the emission of multiple metabolic vasodilators by parenchymal cells. We have proposed a mechanism of regulation, built from well‐known components, which assumes that arterioles are normally dilated in metabolically active tissues, due to the emission of NO by the endothelium of microvessels. Regulation of local blood flow aims at preventing an excessive supply of oxygen (O2) and glucose to the tissue and thus provides an adequate supply, in contrast to the metabolic regulation theory which requires permanent hypoxia to generate the metabolic vasodilators. The mediator of the restrictive signal is superoxide anion (O2−) released by membrane NAD(P)H oxidases into the interstitial space, where it neutralizes NO at a diffusion‐limited rate. This model predicts that the onset of muscle contraction will lead to the cessation of O2− production, which will cause an elevation of interstitial NO concentration and an increase in fluorescence of the NO probe DAF‐FM after its conversion to DAF‐T. The time course of DAF‐T fluorescence in contracting muscle is predicted by also considering the washout from the muscle of the interstitially loaded NO indicator. Experiments using pulse fluorimetry confirmed an increase in the interstitial concentration of NO available for reaction with DAF‐FM during bouts of muscle contraction. The sharp increase in interstitial [NO] is consistent with the hypothesis that the termination of the neutralizing superoxide flow into the interstitium is associated with the activation of mitochondria and a reduction of the interstitial oxygen tension. The advantage of the new model is its ability to explain the interaction of metabolic activity and local blood flow through the adequate delivery of glucose and oxygen.