Objectives The environmental pollutant 2-nitrotoluene (2-NO 2 -T) is carcinogenic and reproductively toxic in animals. In this study, we elucidated the mechanisms of its carcinogenicity and reproductive toxicity. Methods We examined DNA damage induced by 2-NO 2 -T and its metabolite, 2-nitrosotoluene (2-NO-T), using 32 P-5 0 -end-labeled DNA. We measured 8-oxo-7, 8-dihydro-2 0 -deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA and cellular DNA in cultured human leukemia (HL-60) cells treated with 2-NO 2 -T and 2-NO-T. 8-Oxoguanine DNA glycosylase (OGG1) gene expression in HL-60 cells was measured by real-time polymerase chain reaction (PCR). We examined estrogenic activity using an E-screen assay and a surface plasmon resonance (SPR) sensor.Results In experiments with isolated DNA fragments, 2-NO-T induced oxidative DNA damage in the presence of Cu (II) and b-nicotinamide adenine dinucleotide disodium salt (reduced form) (NADH), while 2-NO 2 -T did not. 2-NO-T significantly increased levels of 8-oxodG in HL-60 cells. Real-time polymerase chain reaction (PCR) analysis revealed upregulation of OGG1 gene expression induced by 2-NO-T. An E-screen assay using the human breast cancer cell line MCF-7 revealed that 2-NO 2 -T induced estrogen-dependent cell proliferation. In contrast, 2-NO-T decreased the cell number and suppressed 17b-estradiolinduced cell proliferation. The data obtained with the SPR sensor using estrogen receptor a and the estrogen response element supported the results of the E-screen assay. Conclusions Oxidative DNA damage caused by 2-NO-T and estrogen-disrupting effects caused by 2-NO 2 -T and 2-NO-T may play a role in the reproductive toxicity and carcinogenicity of these entities.