--- - |2 Abstract Endothelium‐dependent responses were first demonstrated 40 years ago in the aorta. Since then, extensive research has been conducted in vitro using conductance vessels and materials derived from them. However, the microcirculation controls blood flow to vital organs and has been the focus of in vivo studies of endothelium‐dependent dilation beginning immediately after the first in vitro report. Initial in vivo studies employed a light/dye technique for selectively damaging the endothelium to unequivocally prove, in vivo, the existence of endothelium‐dependent dilation and in the microvasculature. Endothelium‐dependent constriction was similarly proven. Endothelium‐dependent agonists include acetylcholine (ACh), bradykinin, arachidonic acid, calcium ionophore A‐23187, calcitonin gene‐related peptide (CGRP), serotonin, histamine and endothelin‐1. Normal and disease states have been studied. Endothelial nitric oxide synthase, cyclooxygenase and cytochrome P450 have been shown to generate the mediators of the responses. Some of the key enzyme systems generate reactive oxygen species (ROS) like superoxide which may prevent EDR. However, one ROS, namely H2O2, is one of a number of hyperpolarizing factors that cause dilation initiated by endothelium. Depending upon microvascular bed, a single agonist may use different pathways to elicit an endothelium‐dependent response. Interpretation of studies using inhibitors of eNOS is complicated by the fact that these inhibitors may also inhibit ATP‐sensitive potassium channels. Other in vivo observations of brain arterioles failed to establish nitric oxide as the mediator of responses elicited by CGRP or by ACh and suggest that a nitrosothiol may be a better fit for the latter. - Acta Physiologica, Volume 224, Issue 2, October 2018.