Cyclic AMP (cAMP) is compartmentalised into independent signalling domains within cells, and a focus of current research is elucidating how cAMP is regulated at these intracellular domains. In some compartments, which are near to the plasma membrane, cAMP is generated by transmembrane adenylyl cyclase, but in compartments that are distant from the plasma membrane, such as the mitochondrial matrix, the only source of cAMP is soluble adenylyl cyclase (sAC). Giovanni Manfredi and colleagues previously showed that sAC regulates oxidative phosphorylation (OXPHOS) in the mitochondrial matrix, and now (p. 3713), they study the role of sAC in distinct intracellular domains. The authors examine sAC-knockout fibroblasts and show a defect in OXPHOS activity, accompanied by a phosphorylated (P)-AMPK-driven compensatory increase in OXPHOS components. When enzymatically active sAC is targeted exclusively to the mitochondrial matrix (mt-sAC) in sAC-knockout fibroblasts, P-AMPK levels and OXPHOS function are restored. Next, the authors show that both inositol 3-phosphate receptor (IP3R)-mediated ER Ca2+ release and ER–mitochondria Ca2+ transfer are altered in sAC-knockout fibroblasts. The mechanism underlying defective ER Ca2+ release in sAC-knockout cells is decreased IP3R phosphorylation owing to lack of cAMP signalling. Expression of recombinant sAC in the cytosol corrects ER Ca2+ release, but mt-sAC fails to do so, demonstrating the existence of functionally distinct intracellular sAC compartments. Nevertheless, decreased ER Ca2+ release negatively impacts upon mitochondrial respiration by reducing mitochondrial Ca2+ uptake, which indicates that cytosolic sAC can indirectly affect mitochondrial function through the regulation of ER Ca2+ release. Taken together, these findings demonstrate independent sAC-defined intracellular cAMP signalling domains in the mitochondria and at the IP3R on the ER.