Abstract3‐Nitrobenzanthrone (3‐NBA) is a mutagen and suspected human carcinogen detected in diesel exhaust, airborne particulate matter, and urban soil. We investigated the tissue specific mutagenicity of 3‐NBA at the lacZ locus of transgenic Muta™Mouse following acute single dose or 28‐day repeated‐dose oral administration. In the acute high dose (50 mg/kg) exposure, increased lacZ mutant frequency was observed in bone marrow and colonic epithelium, but not in liver and bladder. In the repeated‐dose study, a dose‐dependent increase in lacZ mutant frequency was observed in bone marrow and liver (2‐ and 4‐fold increase above control), but not in lung or intestinal epithelium. In addition, a concentration‐dependent increase in mutant frequency (8.5‐fold above control) was observed for Muta™Mouse FE1 lung epithelial cells exposed in vitro. 1‐Nitropyrene reductase, 3‐NBA reductase, and acetyltransferase activities were measured in a variety of Muta™Mouse specimens in an effort to link metabolic activation and mutagenicity. High 3‐NBA nitroreductase activities were observed in lung, liver, colon and bladder, and detectable N‐acetyltransferase activities were found in all tissues except bone marrow. The relatively high 3‐NBA nitroreductase activity in Muta™Mouse tissues, as compared with those in Salmonella TA98 and TA100, suggests that 3‐NBA is readily reduced and activated in vivo. High 3‐NBA nitroreductase levels in liver and colon are consistent with the elevated lacZ mutant frequency values, and previously noted inductions of hepatic DNA adducts. Despite an absence of induced lacZ mutations, the highest 3‐NBA reductase activity was detected in lung. Further studies are warranted, especially following inhalation or intratracheal exposures. Environ. Mol. Mutagen., 2008. Published 2008 Wiley‐Liss, Inc.