Firefly luciferase catalyzes the emission of light from luciferin in the presence of oxygen and adenosine triphosphate. This bioluminescence is commonly employed in imaging mode to monitor tumor growth and treatment responses in vivo. A potential concern is that, since solid tumors are often hypoxic, either constitutively and/or as a result of treatment, the oxygen available for the bioluminescence reaction could be reduced to limiting levels, leading to underestimation of the actual number of luciferase-labeled cells during in vivo experiments. We present studies of the oxygen dependence of bioluminescence in vitro in rat 9 L gliosarcoma cells tagged with the firefly luciferase gene (9L^luc). We demonstrate that the bioluminescence signal decreases at pO₂ ⩽ 5%, falling by approximately 50% at 0.2% pO₂. Further experiments showed that the critical threshold for the initiation of metabolic depression in these cells was around 5%. Below this level, the decrease of oxygen saturation was followed by a decrease in intracellular ATP due to the reduction of mitochondrial membrane potential. Hence, the data suggest that the decrease of intracellular ATP level in vitro is the limiting factor for bioluminescence reaction and so is responsible for the reduction of bioluminescence signal in 9L^luc cells in acute hypoxia, rather than luciferase expression or oxygen itself.