Second messenger signaling between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) is poorly understood, but intracellular Ca2+ concentrations ([Ca2+]i) in the 2 cells are coordinated, possibly through gap junctions at the myoendothelial junction. To study heterocellular calcium signaling, we used a vascular cell coculture model composed of monolayers of ECs and VSMCs. Stimulation of either cell type leads to an increase in [Ca2+]i in the stimulated cell and a secondary increase in [Ca2+]i in the other cell type that was blocked by gap junction inhibitors. To determine which second messengers are involved, we initially depleted Ca2+ stores in the endoplasmic reticulum Ca2+ with thapsigargin in ECs or VSMCs, but this had no effect on heterocellular calcium signaling. Alternatively, we loaded ECs or VSMCs with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) to buffer changes in [Ca2+]i. BAPTA loading of ECs inhibited agonist-induced increases in intracellular calcium concentration ([Ca2+]i), in both ECs and VSMCs. In contrast, BAPTA loading of the VSMCs blunted the VSMC response but did not alter the secondary increase in EC [Ca2+]i. Xestospongin C (an inositol 1,4,5-trisphosphate receptor inhibitor) had no effect on the secondary Ca2+ response, but when xestospongin C or thapsigargin was loaded into ECs and BAPTA into VSMCs, intercellular Ca2+ signaling was completely blocked. We conclude that 1,4,5-trisphosphate and Ca2+ originating in the VSMCs induces the secondary increase in EC [Ca2+]i but stimulation of the ECs generates a Ca2+ dependent response in the VSMCs.