The role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca²⁺ signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells ([Ca²⁺]_o = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) plus -hydroxy-butyrate (HB), or iodoacetic acid (IAA) caused an increase of [Ca²⁺]_i. CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca²⁺]_i. The rise of [Ca²⁺]_i was characterized by a transient spike followed by a small sustained plateau of elevated [Ca²⁺]_i. In the absence of extracellular Ca²⁺ 2-DG caused an increase in [Ca²⁺]_i suggesting that inhibition of glycolysis directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. The inositol-1,4,5-trisphosphate receptor (IP₃R) inhibitor 2-APB abolished the KN-93- and 2-DG-induced Ca²⁺ response. Ca²⁺ release was initiated in peripheral cytoplasmic processes from where activation propagated as a [Ca²⁺]_i wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca²⁺]_i. Propagating [Ca²⁺]_i waves were preceded by [Ca²⁺]_i oscillations and small, highly localized elevations of [Ca²⁺]_i (Ca²⁺ puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca²⁺ response, and vice versa during inhibition of CaMKII 2-DG-induced Ca²⁺ release was attenuated. Similar results were obtained with pyr+HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of [Mg²⁺]_i, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca²⁺ release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP₃R.