To elucidate how tumor cells produce energy in oxygen-depleted microenvironments, we studied the possibility of mitochondrial electron transport without oxygen. We produced well-controlled oxygen gradients (DeltaO2) in monolayer-cultured cells. We then visualized oxygen levels and mitochondrial membrane potential (DeltaPhim) in individual cells by using the red shift of green fluorescent protein (GFP) fluorescence and a cationic fluorescent dye, respectively. In this two-dimensional tissue model, DeltaPhim was abolished in cells >500 mum from the oxygen source [the anoxic front (AF)], indicating limitations in diffusional oxygen delivery. This result perfectly matched GFP-determined DeltaO2. In cells pretreated with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase domain-containing protein (PHD) inhibitor, the AF was expanded to 1,500-2,000 mum from the source. In these cells, tissue DeltaO2 was substantially decreased, indicating that PHD pathway activation suppressed mitochondrial respiration. The expansion of the AF and the reduction of DeltaO2 were much more prominent in a cancer cell line (Hep3B) than in the equivalent fibroblast-like cell line (COS-7). Hence, the results indicate that PHD pathway-activated cells can sustain DeltaPhim, despite significantly decreased electron flux to complex IV. Complex II inhibition abolished the effect of DMOG in expanding the AF, although tissue DeltaO2 remained shallow. Separate experiments demonstrated that complex II plays a substantial role in sustaining DeltaPhim in DMOG-pretreated Hep3B cells with complex III inhibition. From these results, we conclude that PHD pathway activation can sustain DeltaPhim in an otherwise anoxic microenvironment by decreasing tissue DeltaO2 while activating oxygen-independent electron transport in mitochondria.