Purpose: PR-104, a bioreductive prodrug in clinical trial, is a phosphate ester which is rapidly metabolized to the corresponding alcohol PR-104A. This dinitrobenzamide mustard is activated by reduction to hydroxylamine (PR-104H) and amine (PR-104M) metabolites selectively in hypoxic cells, and also independently of hypoxia by aldo-keto reductase (AKR) 1C3 in some tumors. Here, we evaluate reductive metabolism of PR-104A in mice and its significance for host toxicity. Methods: The pharmacokinetics of PR-104, PR-104A and its reduced metabolites were investigated in plasma and tissues of mice (with and without SiHa or H460 tumor xenografts) and effects of potential oxidoreductase inhibitors were evaluated. Results: Pharmacokinetic studies identified extensive non-tumor reduction of PR-104A to the 5-amine PR-104H (identity of which was confirmed by chemical synthesis), especially in liver. However, high concentrations of PR-104H in tumors that suggested intra-tumor activation is also significant. The tissue distribution of PR-104M/H was broadly consistent with the target organ toxicities of PR-104 (bone marrow, intestines and liver). Surprisingly, hepatic nitroreduction was not enhanced when the liver was made more hypoxic by hepatic artery ligation or breathing of 10 oxygen. A screen of non-steroidal anti-inflammatory drugs identified naproxen as an effective AKR1C3 inhibitor in human tumor cell cultures and xenografts, suggesting its potential use to ameliorate PR-104 toxicity in patients. However, neither naproxen nor the pan-CYP inhibitor 1-aminobenzotriazole inhibited normal tissue reduction of PR-104A in mice. Conclusions: PR-104 is extensively reduced in mouse tissues, apparently via oxygen-independent two-electron reduction, with a tissue distribution that broadly reflects toxicity.