The popular use of in vitro systems for toxicity studies has increased dramatically over the past decade. Among the in vitro systems used, primary hepatocyte cultures are the most widely employed. However, in addition to being difficult to obtain and maintain in culture, the functional heterogeneity of liver is absent. Primary organ cultures of thin liver slices should overcome these limitations but the lack of a reproducible method for the rapid preparation of thin, consistent slices, combined with the difficulty in maintaining adult liver tissue in culture, has hindered their use for in vitro hepatotoxicity studies. Using a recently-developed tissue slicer, thin, consistent liver slices were prepared rapidly under minimally traumatic conditions. Subsequent culture of these slices in a novel dynamic organ culture system (DOCS) resulted in a maintenance of hepatocyte functional integrity. Slice adenosine triphosphate (ATP) and K⁺ content were maintained at in vivo levels, following an initial recovery period (2-4h) for up to 20h. Protein synthesis and secretion were linear for 20h and 16h respectively. Slices also synthesized glycogen between 4 and 12h in culture and were hormonally-responsive during the 20h culture period as demonstrated by a two-fold stimulation of glucose production by glucagon (10⁻⁷ M). Bromobenzene and allyl alcohol hepatotoxicity were studied in this system of organ culture. The slices retained their biotransformation ability for at least 6h based on maintenance of cytochrome P-450 content and O-deethylase activity. Either compound caused dose (.01-1.0 mM) and time (0-6h) dependent cytotoxicity as indicated by the loss of slice K⁺, inhibition of protein synthesis and leakage of lactate dehydrogenase (LDH). By 2h, a significant (p < 0.05) inhibition of protein synthesis was observed in allyl alcohol (.05 mM) treated slices. At 4h and 6h, significant loss of slice K('+), LDH, and inhibition of protein synthesis were evident in slices exposed to allyl alcohol (0.25 mM) or bromobenzene (0.5 mM). This toxicity was blocked by co-treatment with pyrazole (1.0 mM) or SKF 525-A (100 μM) in slices exposed to allyl alcohol or bromobenzene, respectively. Therefore, this system provides a new tool for the in vitro study of hepatotoxicity under conditions where hepatocellular functional integrity and biotransformation are maintained.