A capillary zone electrophoresis-based method to evaluate the cytotoxicity of substances to Caco-2 cells was established. The estimation of the injected cell number (500-5000) and the minor effect of injection condition on cytotoxicity determination were investigated. Caco-2 cells the best model of the intestinal absorptive epithelium, were treated with substances and then stained with Trypan Blue and fixed with paraformaldehyde. The treated Caco-2 cells were detected simultaneously at 590 nm and 214 nm, and the absorbance ratio of the two wavelengths (R-590/214) can reflect simultaneously the loss of cell membrane integrity and the degradation/leak of intracellular components and indicate the cytotoxicity of substances. The cytotoxicity of the four substances sodium sulfite (Na2SO3), methyl mercury (MeHg), paclitaxel (PTX), and cadmium chloride (CdCl2) were determined and compared. There was no obvious cytotoxicity caused by 20 mu M Na2SO3 for 24 h treatment, and the toxicity of the other three toxicants was sequenced as: CdCl2 > MeHg > PTX. The results are in good agreement with the references and the conventional Trypan Blue exclusion counting assay.