To study endothelial cell (EC)– specific Ca²⁺ signaling in vivo we engineered transgenic mice in which the Ca²⁺ sensor GCaMP2 is placed under control of endogenous connexin40 (Cx40) transcription regulatory elements within a bacterial artificial chromosome (BAC), resulting in high sensor expression in arterial ECs, atrial myocytes, and cardiac Purkinje fibers. High signal/noise Ca²⁺ signals were obtained in Cx40^BAC-GCaMP2 mice within the ventricular Purkinje cell network in vitro and in ECs of cremaster muscle arterioles in vivo. Microiontophoresis of acetylcholine (ACh) onto arterioles triggered a transient increase in EC Ca²⁺ fluorescence that propagated along the arteriole with an initial velocity of 116 µm/s (n=28) and decayed over distances up to 974 µm. The local rise in EC Ca²⁺ was followed (delay, 830±60 ms; n=8) by vasodilation that conducted rapidly (mm/s), bidirectionally, and into branches for distances exceeding 1 mm. At intermediate distances (300 to 600 µm), rapidly-conducted vasodilation occurred without changing EC Ca²⁺, and additional dilation occurred after arrival of a Ca²⁺ wave. In contrast, focal delivery of sodium nitroprusside evoked similar local dilations without Ca²⁺ signaling or conduction. We conclude that in vivo responses to ACh in arterioles consists of 2 phases: (1) a rapidly-conducted vasodilation initiated by a local rise in EC Ca²⁺ but independent of EC Ca²⁺ signaling at remote sites; and (2) a slower complementary dilation associated with a Ca²⁺ wave that propagates along the endothelium.