The cell viability assay using cultured cells is of great advantage to elucidate the biological effect of potentially toxic substances. Recently, a novel assay system, Tetracolor One cell proliferation assay (Seikagaku Co., Tokyo, Japan), has been developed. In this report, we compare the results of the Tetracolor One assay regarding the cytotoxic effect of three heavy metal salts on cultured adult keratinocytes to those of the neutral red dye uptake assay and the MTT eluted stain assay. In this study, these three methods showed almost similar results. Compared to the other two methods, however, the Tetracolor One assay, which requires only one-step procedure before spectrophotometric measurement, is easier to use, and errors in measurement, which may be produced through the multistep procedure, are much less in this assay. Therefore, we believe that the Tetracolor One assay system is useful for assessing the cytotoxic effect of heavy metals on cultured human keratinocytes.