Voltage-gated calcium channels are involved in the excitation-contraction mechanism of resistance vessels. However, T-type channels are also involved in relaxation of mesenteric, coronary and renal blood vessels. We investigated the mechanisms involved in the T-type channel-dependent dilatation. The secondary dilatation after potassium-induced contraction of isolated perfused mesenteric arteries was reduced significantly in Cav3.1-/- compared to Wt mice and use of eNOS -/- mice led to a significantly attenuated dilatation. Western blotting showed no difference in phosphorylated eNOS-pS1117 levels between wild type and Cav3.1-/- mice. Immunoprecipitation of Cav3.1 pulled down eNOS, and immunoprecipitation of eNOS pulled down Cav3.1 showing co-localization of Cav3.1 and eNOS. Confocal laser-scanning microscopy of mesenteric arteries labeled with antibodies against eNOS and Cav3.1 confirmed the co-localization of eNOS and Cav3.1 in endothelial cells, and showed that Cav3.1was also expressed in vascular smooth muscle cells. The basal cGMP levels in isolated aortae were not different between genotypes. We conclude that endothelial calcium-influx through Cav3.1 T-type calcium channels that are co-localized with eNOS, stimulates eNOS and NO production which, in turn, affects NO-dependent dilatation after depolarization in mesenteric resistance arteries. This mechanism could be important for maintaining the systemic impact of NO on total peripheral resistance.