The endothelium modulates the contractile state of the smooth muscle by the release of vasodilator autacoids. These include nitric oxide (NO), prostaglandins and a third, currently not well characterized dilator principle which acts through hyperpolarization of the smooth muscle and is therefore termed endothelium-derived hyperpolarizing factor (EDHF). Different chemical substances have been identified to be released from the endothelium and induce smooth muscle hyperpolarization in different vascular beds, e.g. K+ ions, epoxyeicosanoids, and hydrogen peroxide, and may therefore account for an EDHF. However, recently it was suggested that the endothelial hyperpolarization is directly transferred through myoendothelial gap junctions (MEGJ) to the adjacent smooth muscle without requiring a diffusable factor. The evidence for this was obtained mostly in isolated vessels studied in vitro. Under these conditions, EDHF-type dilations were abrogated after blockade of the gap junction protein connexin40 (Cx40) using a specific antibody loaded into endothelial cells. However, in the cremaster microcirculation studied in vivo EDHF-type dilations remain fully intact in Cx40-deficient mice. In contrast, EDHF-type dilations were completely abrogated in a small artery supplying the skeletal muscle (A. gracilis) in Cx40-deficient mice studied in vitro using wire-myography. However, if exactly this vessel was investigated in vivo, EDHF-type dilations were unhindered in Cx40-deficient mice and not different from wildtype controls as observed in cremasteric arterioles studied by intravital microscopy. Therefore, we suggest that myoendothelial coupling is only contributing to EDHF-type dilations under conditions in vitro such as wire myography because this technique leads to the loss of a potent, but distinct EDHF-type dilator pathway. This dilator principle completely accounts for EDHF-type dilations in vivo and acts independent of the gap junction protein Cx40.