Ever since nitric oxide (NO) was discoverd to be a powerful endothelium-derived vasorelaxing substance, a number of methods have been developed to investigate the role of NO in the vasodilatory process in humans. The present review discusses the advantages and limitations of the three most commonly used techniques to assess endothelium-dependent vasodilation (EDV) in the coronary circulation, the brachial artery, and in the forearm circulation. Furthermore, six less established methods for the evaluation of EDV in different vascular beds in humans are presented.