HC Orange No. 1 (HCO1, 2-nitro-4'-hydroxydiphenylamine) (CAS No. 54381-08-7) is used as a colorant in commercial hair dyes. The cytotoxicity of HCO1 to L929 mouse connective tissue fibroblast cells was determined by use of a battery of endpoints that included electron transport activity determined by the MTT assay and fluorescence microscopy with Hoechst 33258, DNA agarose gel electrophoresis detected by ethidium bromide and flow cytometry. Cytotoxicity of HCO1 was found to be concentration- and time-dependent with EC(50) values of 28.0 and 12.0mg/L after 24h or 48h exposure, respectively. The mechanism of toxicity of HCO1 was more characteristic of necrosis than apoptosis. These results can be used to evaluate the relative in vitro toxicity of other dyes and to elucidate their mechanisms of toxicity so that accurate assessment of risks to humans can be made, especially from dermal exposure.