Conventional in vitro cytotoxicity tests usually do not include toxicokinetic processes that affect final toxicity in the entire body. To overcome this limitation, we have been developing several types of new toxicity test systems and applying them to evaluate hazardous chemicals or environmental samples. In this review, we described two of these new systems; one is a batch-type gas exposure system based on air-liquid interface culture of lung epithelial cells, and the other is a simple double-layered coculture system incorporating permeation and biotransformation processes occurring in the small intestine. In addition, we introduce our latest approach toward further miniaturization of existing tests, that is, determination of minimum cell number necessary for obtaining physiologically-relevant tissue responses.