Nitric oxide (NO) modulates cellular metabolism by competitively inhibiting the reduction of O₂ at respiratory complex IV. The aim of this study was to determine if this effect could enhance cell survival in the hypoxic solid tumour core by inducing a state of metabolic arrest in cancer cells. Mitochondria from human alveolar-type II-like adenocarcinoma (A549) cells showed a 4-fold increase in NO-sensitive 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) fluorescence and 6-fold increase in Ca²⁺-insensitive NO synthase (NOS) activity during equilibration from PO₂s of 10023mmHg which was abolished by N-nitro-L-arginine methyl ester-HCl (L-NAME) and the inducible (i)NOS inhibitor, N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL). Similarly, cytosolic and compartmented DAF-FM fluorescence increased in intact cells during a transition between ambient PO₂ and 23mmHg and was abolished by transfection with iNOS antisense oligonucleotides (AS-ODN). In parallel, mitochondrial membrane potential (_m), measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), decreased to a lower steady state in hypoxia without change in glycolytic rate, adenylate energy charge or cell viability. However, L-NAME or iNOS AS-ODN treatment maintained _m at normoxic levels irrespective of hypoxia and caused a marked activation of glycolysis, destabilisation energy charge and cell death. Comparison with other cancer-derived (H441) or native tissue-derived (HBE; alveolar type II) lung epithelial cells revealed that the hypoxic suppression of _m was common to cells that expressed iNOS. The controlled dissipation of _m, absence of an overt glycolytic activation and conservation of viability suggest that A549 cells enter a state of metabolic suppression in hypoxia which inherently depends upon the activation of iNOS as PO₂ falls.