A new technique for microplate assay of cell viability using a tetrazolium compound. We have developed a simple method for evaluating the relative viability of cultured mammalian cells using a tetrazolium compound, XTT. The principle of this method is as follows. After culturing cells in a microtiter plate (in the case of 96-well microplates), the medium is removed from the microplate and the cells are treated with a tetrazolium compound and a riboflavin solution, which are contained in the medium used for culturing human melanoma A375 cells. Then the cell viability can be estimated from the amount of yellowish color formed in the microplate, without the addition of a supravital dye. The relative viability of A375 cells could be estimated with this method, based on the relationship between the viability and absorbance (O.D.) using a microplate reader (SpectraMax M5). The number of A375 cells, which had grown in Dulbecco's Modified Eagle's Medium (DMEM), was determined to be 2.6 x 10(4) in one microplate using this method. These results were almost consistent with the results determined with a CCK-8 (Cell Counting Kit-8) kit which is another widely used method for estimating the relative viability. Thus, the relative viability of cells could be determined with the XTT method in this study, without the use of expensive reagents. Furthermore, a comparison of the results between XTT and CCK-8 suggested that the relative cell viabilities could be determined more precisely with the XTT method than with the CCK-8 method. Therefore, the XTT method may be a more useful alternative method than the CCK-8 method for microplate assay of cell viability.