It is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation in O2-sensing cells, and thereby mediates, in part, cell activation. However, the mechanism by which this process is coupled to discrete, cell-specific Ca2+ signalling mechanisms remains elusive. We considered the possibility that hypoxia may increase the cellular ATP/AMP ratio, increase the activity of AMP-activated protein kinase (AMPK) and thereby evoke Ca2+ signals in O2-sensing cells. Co-immunoprecipitation identified α1β2γ1 as the primary AMPK isozyme in pulmonary arterial smooth muscle, whilst the tissue-specific distribution of AMPK activities and their activation by hypoxia suggested that the AMPK-α1 catalytic subunit isoform is key to the regulation of O2-sensing cells. Strikingly, 3D reconstruction of immuno- fluorescence images showed AMPK-α1 to be located throughout the cytoplasm of pulmonary arterial smooth muscle cells and, by contrast, targeted to the plasma membrane in carotid body glomus cells. Consistent with these observations Ca2+imaging, tension recording and electrophysiology demonstrated that AMPK, like hypoxia, activates each cell type via discrete Ca2+signalling mechanisms: cyclic ADP-ribose-dependent Ca2+ mobilization from the sarcoplasmic reticulum via ryanodine receptors in pulmonary arterial smooth muscle cells and voltage-gated Ca2+ influx into carotid body glomus cells. Thus, metabolic-sensing by AMPK underpins the cell-specific response of O2-sensing cells to hypoxia.