The cytotoxic effects of ethanol on the colony forming efficiency of PLC/PRF/5 human hepatoma cells were investigated. When ceils were inoculated into plastic dishes and incubated in a CO2 incubator (open system), no cytotoxicity was observed with concentrations of up to 1% of ethanol in the culture medium. However, when flasks were used and their caps were tightly closed (closed system), some cytotoxicity was demonstrated, the lethal dose of ethanol for 50% cell death being 0.64 + 0.04%. The contrasting results in the open and closed systems were due to the evaporation of ethanol, since ethanol was very rapidly lost from the culture medium under the open system, whereas there was no substantial loss of ethanol under the closed system. The present results indicate that a closed system should be used to evaluate cytotoxic effects of volatile substances such as ethanol.