Vascular coordination in the microcirculation depends on gap junctional intercellular communication (GJIC) which is reflected by the conduction of locally initiated vasomotor responses. However, little is known about the regulation of GJIC in vivo. We hypothesized that endothelial NO regulates GJIC and therefore studied whether conduction of constrictions and dilations along the vessel wall is modulated by modifying the level of microcirculatory NO. Arterioles were focally stimulated using high K⁺ or acetylcholine in the cremaster muscle in situ and diameter changes were assessed at the local and remote upstream sites by intravital microscopy. Local stimulation with K+ initiated a constriction which conducted along the arteriole with diminishing amplitude (length constant : 371±42µm). After L-nitro-arginine (LNA), increased to 507±30µm indicating that GJIC is attenuated by endogenous NO. Exogenous NO, but not adenosine, reduced after LNA in a reversible, concentration-dependent and mainly cGMP-dependent manner as assessed by inhibition of soluble guanylate cyclase. In eNOS-deficient mice, was 530±80µm and thus similar to wildtype mice after LNA. Exogenous NO likewise reduced in these mice. The effects of NO were comparable to wildtype animals in Cx40-deficient mice which excludes Cx40 as a specific target of NO. In contrast to constrictions, the amplitude of conducted dilations upon acetylcholine did not diminish up to 1300µm and were not altered by LNA or exogenous NO. We conclude that endogenously released NO attenuates the conduction of vasoconstrictions most likely due to a modulation of GJ conductivity. We suggest that this effect is specific for smooth muscle cells which probably transmit constricting signals and involves other connexins than Cx40. This mechanism might support the dilatory potency of NO by preventing the conduction of remote vasoconstrictions into areas with basal or activated NO release.