Aflatoxin B1 (AFB1) caused marked, rapid (1 h) inhibition of RNA synthesis and subsequent cytotoxic response in isolated and primary cultured hepatocytes, from control rats, which are known to metabolise AFB1. A rat liver-derived cell line, BL8L, was much less susceptible to these effects of AFB1. These cells have no detectable AFB1 metabolising capacity, but a less potent, anti-mitotic action of AFB1 was observed in the BL8L cell line. Thus AFB1 would seem to require metabolism to exert its acute cytotoxic action which is found at very low AFB1 concentrations, although a direct antimitotic effect, independent of metabolism, is seen in dividing cells. Phenobarbitone and 3-methylcholanthrene in vivo pretreatments, known inducers of AFB1 metabolism, resulted in reduced AFB1 inhibition of RNA synthesis and cytotoxicity in hepatocytes, but only at lower concentrations of AFB1 used, whereas cells from AFB1 fed rats were much less susceptible to AFB1 toxicity at all concentrations used. This resistance to cytotoxicity of AFB1 would appear to involve detoxification mechanisms, primarily the formation of polar conjugates of AFB1 metabolites, particularly glutathione conjugates. These cell culture systems are useful for studying association between metabolism and cytotoxicity of AFB1, and other xenobiotics.