o-Aminoazotoluene (AAT) has been evaluated as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). The Ames test found it to be mutagenic in the presence of a metabolic activation system, whereas it has little clastogenicity either in vitro or in vivo in the chromosomal aberration assay. AAT is also carcinogenic in the lung or liver of mice and rats given long-term administrations. Therefore, metabolites generated in the liver etc. may have gene mutation activity, and carcinogenesis would occur. We examined the mutagenicity of AAT in a gene mutation assay, using lacZ transgenic mice (MutaMice) and a positive selection method. AAT showed positive results for organs with metabolic functions, such as liver and colon and other organs. Positive results were also seen in an Ames test in the presence of metabolic activation and negative results seen in a chromosomal aberration test. Therefore, AAT had the potential to cause gene mutation in the presence of metabolic activation systems in vitro and the same reaction was confirmed in vivo with organs with metabolic function, such as liver and colon, but little clastogenicity in vitro or in vivo. Thus, metabolites with gene mutation activity may be responsible for the carcinogenicity of AAT. The transgenic mouse mutation assay proved to be useful for concurrent assessment of in vivo mutagenicity in multiple organs and to supplement the standard in vivo genotoxicity tests, such as the micronucleus assay which is limited to bone marrow as the only target organ.