The transcription factor hypoxia-inducible factor-1 (HIF-1) is critical for erythropoietin and other factors involved in the adaptation of the organism to hypoxic stress. Conflicting results have been published regarding the role of the mitochondrial electron transport chain (ETC) in the regulation of HIF-1alpha. We assessed cellular hypoxia by pimonidazole staining and blotting of the O2-labile HIF-1 alpha-subunit in human osteosarcoma cell cultures (U2OS and 143B). In conventional, gas-impermeable cell culture dishes, ETC inhibitors had no effect on pimonidazole staining or HIF-1alpha abundance in a 20% O2 atmosphere; both parameters were undetectable. Pimonidazole staining and HIF activity were substantial in 0.1% O2 irrespective of ETC inhibition. At an intermediate oxygen concentration (3% O2) pimonidazole staining and HIF-alpha expression were detectable but strongly reduced after ETC inhibition in conventional cell cultures. All effects of ETC inhibition on HIF-1alpha regulation were eliminated in gas-permeable dishes. As shown in a 143B subclone deficient in mitochondrial DNA (206rho0), genetic inactivation of the ETC led to similar responses with respect to HIF-1alpha regulation as ETC inhibitors. Our data demonstrate that reduction of oxygen consumption reduces the O2 gradient in conventional cell cultures, causing elevation of the cellular O2 concentration, which leads to degradation of HIF-alpha.