We present an improved cytotoxicity test for reactive metabolites, in which the 59 microsomal fraction of rat liver homogenate is encapsulated in alginate gel microbeads to avoid cytotoxic effects of 59-self-generated toxicants, microsomal lipid peroxides. The 59-encapsulated gel microbeads were prepared by a coaxial two-fluid nozzle and surfaces of the microbeads were coated with poly-L-lysine (PLL). Although the initial metabolic rate of the 59-encapsulated gel microbeads was about 20% slower than that of bare 59, the microbeads prevented the leakage of microsomal lipid peroxides thanks to the dense alginate and PLL polymer networks. In fact, the half maximal effective concentration of the indirect mutagen cyclophosphamide on NIH3T3 cells in the presence of the 59-encapsulated gel microbeads was about 5 times higher than that in the presence of bare S9. Use of the S9-encapsulated gel microbeads enabled the more accurate evaluation of the cytotoxicity of the reactive metabolites without the S9-based cytotoxicity. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.