Hypoxia in tumor tissues plays a pivotal role in tumor progression and angiogenesis, and is associated with cancer therapeutic resistance. For the diagnosis of hypoxia, non-invasive imaging techniques, especially positron emission tomography (PET) with 2-nitroimidazole-based probes, are used, since 2-nitroimidazole-based probes are considered to undergo reductive metabolism on their 2-nitroimidazole moiety and become trapped in hypoxic cells. However, the detailed mechanism of their accumulation remains unclear because of the difficulty in estimating the metabolites by radioisotopic analysis. Imaging mass spectrometry (IMS) can distinguish the distribution patterns of the drug and its metabolites. To clarify the accumulation mechanism of 2-nitroimidazole-based probes in hypoxic cells, we evaluated [¹⁸F]fluoromisonidazole (FMISO), a 2-nitroimidazole-based PET probe, in combination with radioisotopic analysis and IMS. We found that the glutathione conjugate of reduced FMISO (amino-FMISO-GS) was the main FMISO metabolite, and was specifically distributed in the hypoxic regions of tumors. The same phenomenon was observed when we examined another 2-nitroimidazole-based probe, pimonidazole. The in vitro cellular uptake study revealed that FMISO accumulation in hypoxic cells depends on the cell type. In those cells exhibiting higher FMISO uptake, the reactive glutathione level and enzyme (glutathione S-transferase; GST) activity catalyzing the glutathione conjugation reaction was significantly higher, whereas the expression level of the efflux transporter (multidrug resistance-associated protein 1; MRP1) was significantly lower. Our study suggests that 2-nitroimidazole-based probes accumulate in hypoxic cells via glutathione conjugation following reductive metabolism, which depends not only on the glutathione conjugation capacity of the cells but also on hypoxic conditions.