Hepatocytes from control, phenobarbitone- and β-naphthoflavone-treated rats were cultured for 24 hr and then exposed to seven known hepatotoxins: butylated hydroxytoluene, valproic acid, 2-methylfuran, 4-ipomeanol, 6-thiopurine and precocenes I and II. After a further 24 hr in culture, cell survival was determined by assaying mitochondrial reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. All the compounds, except 4-ipomeanol, were more toxic to hepatocytes cultured from phenobarbitone-treated rats than to hepatocytes from either control or β-naphthoflavone-treated rats. The toxicity of 4-ipomeanol was increased to a similar extent by both inducers. Although to a lesser extent than phenobarbitone, β-naphthoflavone treatment in vivo increased the toxicity in vitro of all the other test compounds except valproic acid and butylated hydroxytoluene. In general the observed changes in toxicity agreed with in vivo data in the literature. When rat hepatocytes were treated in vitro with phenobarbitone or β-naphthoflavone, 4-ipomeanol was not toxic to either control or phenobarbitone-treated cultures, although some toxicity was detected in the β-naphthoflavone-treated cultures. Precocene II was not toxic to the control cultures but was toxic to both phenobarbitone- and β-naphthoflavone-treated cultures. However, as with 4-ipomeanol, the enhancement of toxicity after induction in vitro, particularly with phenobarbitone, was much reduced compared with treatment in vivo. The results suggest that hepatocytes cultured from inducer-treated rats can be used as a model for the investigation of P-450-mediated toxicity in vitro.