Quenched-phosphorescence oxygen (O(2)) sensing technique allows non-invasive, real-time monitoring of both intra- and extracellular O(2) concentration in respiring samples. Using this technique we investigated O(2) gradients in populations of neurosecretory PC12 cells cultured in 96-well plates and exposed to graded hypoxia at rest and upon metabolic stimulation. Under high atmospheric O(2) (10-21%) the respiration of resting cells dictated that local O(2) was moderately reduced, and at a certain threshold (6% in galactose medium) cell layer became practically anoxic. Furthermore, cell stimulation triggered a major redistribution of O(2) and a prominent 'hypoxic overshoot' mediated by diffusion. The deep, prolonged cell deoxygenation upon stimulation was matched by an increase in nuclear HIF-1alpha levels. In the presence of nitric oxide the hypoxic overshoot was truncated and HIF-1alpha stabilization inhibited. Thus, the main determinants which impact upon cellular O(2) levels and oxygen-sensitive signaling pathways are the atmospheric O(2), sample geometry, cell density, respiration rate and its dynamics. Changes in any of these parameters can significantly alter the O(2) levels experienced by the cells and the subsequently activated signaling pathways. This technique, which provides simple and reliable monitoring of cell oxygenation, is therefore important for hypoxia research, metabolic studies and experiments with respiring cells.