There is substantial interest in small molecules that can be used to detect or kill the hypoxic (low oxygen) cells found in solid tumors. Nitroaryl moieties are useful components in the design of hypoxia-selective imaging agents and prodrugs because one-electron reductases can convert the nitroaryl group to nitroso, hydroxylamino, and amino metabolites selectively under low oxygen conditions. Here, we describe the in vitro, cell free metabolism of a pro-fluorescent substrate, 6-nitroquinoline (1) under both aerobic and hypoxic conditions. Both LC-MS and fluorescence spectroscopic analyses provided evidence that the one-electron reducing enzyme system, xanthine/xanthine oxidase, converted the nonfluorescent parent compound 1 to the known fluorophore 6-aminoquinoline (2) selectively under hypoxic conditions. The presumed intermediate in this reduction process, 6-hydroxylaminoquinoline (6), is fluorescent and can be efficiently converted by xanthine/xanthine oxidase to 2 only under hypoxic conditions. This finding provides evidence for multiple oxygen-sensitive steps in the enzymatic conversion of nitroaryl compounds to the corresponding amino derivatives. In a side reaction that is separate from the bioreductive metabolism of 1, xanthine oxidase converted 1 to 6-nitroquinolin-2(1H)-one (5). These studies may enable the use of 1 as a fluorescent substrate for the detection and profiling of one-electron reductases in cell culture or biopsy samples. In addition, the compound may find use as a fluorogenic probe for the detection of hypoxia in tumor models. The occurrence of side products such as 5 in the enzymatic bioreduction of 1 underscores the importance of metabolite identification in the characterization of hypoxia-selective probes and drugs that employ nitroaryl units as oxygen sensors.