Despite extensive study of previously favoured hypotheses, the precise mechanism of chemo-transduction in “O₂–sensing” cells remains obscure (Gonzalez et al., 2002). We now propose a novel mechanism by which hypoxia may affect cell function. Our data are consistent with the view that AMP-activated protein kinase (AMPK; Hardie et al., 2003), which monitors the AMP:ATP ratio as an index of metabolic stress, modulates O₂-sensitive Ca²⁺ signalling mechanisms. Western blotting, immunoprecipitate kinase assays and immunocytochemistry identified AMPK in rat pulmonary artery smooth muscle. AMPK activation by phenformin (10 mM) or AICAR (1 mM) increased the Fura-2 fluorescence ratio (F340/F380) by 0.086 ± 0.017 (n = 7; mean ± S.E.M.) and 0.096 ± 0.009 (n = 22), respectively, in isolated rat pulmonary artery smooth muscle cells (for methods see Boittin et al, 2002). The increase in intracellular Ca²⁺ induced by 1mM AICAR was unaffected by removal of extracellular Ca²⁺ (1mM EGTA; n = 12). However, it was significantly inhibited by depletion of sarcoplasmic reticulum (SR) stores with 10 mM caffeine and 10 μM ryanodine (74%, n = 8, P<0.001, Student's t test) and by blockage of cyclic adenosine diphosphate-ribose (cADPR) with 100 μM 8-bromo-cADPR (83%, n = 23, P<0.0001). Thus, AMPK activation induces cADPR-dependent SR Ca²⁺ release via ryanodine receptors in pulmonary arterial smooth muscle. The effect of AMPK activation on carotid body glomus cells was quite different (isolated as previously described; Wyatt and Peers, 1993). 1mM AICAR increased the F340/F380 ratio (0.07 ± 0.018; n=11) in a manner that was reversed by 100 μM Ca²⁺ and by removal of extracellular Ca²⁺ (n = 6). Thus, AMPK activation in carotid body glomus cells leads to voltage-gated Ca²⁺ influx. We conclude that AMPK mediates cell-specific Ca²⁺ signalling mechanisms in carotid body glomus cells and pulmonary arterial smooth muscle cells in a manner that may be tailored to suit each cells function. Our findings suggest that AMPK may couple cell metabolism and Ca²⁺ signalling. Animals were killed humanely.