1. In rat hepatocyte cultures, clonidine and malotilate were cytotoxic at 170 microM but did not induce changes at lower concentrations during 24 h exposure. Amitriptyline induced cell injury at 20 microM but was ineffective at 2 microM. 2. In co-culture of rat or human hepatocytes with rat liver epithelial cells, 2 microM amitriptyline was cytotoxic after 7 days treatment whereas 70 to 100 microM clonidine or malotilate gave no significant effect. 3. These results suggest that co-cultured hepatocytes which retain their differentiated state for several days or weeks, represents a promising tool for studying hepatotoxicity from chronic treatment in vitro.