The threshold for hepatotoxicity and cholestasis induced by methylene dianiline (DAPM) in rats is between 25 and 75 mg/kg (Bailie et al., Environ. Health Perspect., 124, 25-30, 1993). Our objectives were to determine if a minimally toxic dose of DAPM provided a model system for studies of selective injury to biliary epithelial cells (BEC) in vivo. Thus, we examined the effects of 50 mg DAPM/kg on (1) biliary constituents, (2) liver constituents likely involved in DAPM biotransformation/detoxification, and (3) early morphological and histochemical changes in the liver. Male Sprague Dawley rats had biliary cannulas positioned under pentobarbital anesthesia. After 1 h of control bile collection, rats received 50 mg DAPM/kg po in 35% ethanol or 35% ethanol only. Bile was collected for another 6 h. Histochemical, ultrastructural, and biochemical liver alterations were assessed at 3 h or at 3 and 6 h. DAPM had minimal effects on biliary bile salt and bilirubin excretion over 6 h. Biliary glucose and protein excretion were increased approximately 2-fold starting in Hour 1, while inorganic phosphate excretion was not increased until Hour 2. Biliary glutathione excretion initially increased (Hour 1) but then declined steadily for 5 h. Microsomal cytochrome P-450 activities were transiently decreased at 3 h but had returned to control values by 6 h. Liver glutathione (GSH and GSSG) was not affected by DAPM at 3 or 6 h. Necrosis of intrahepatic bile ducts was severe at 6 h with moderate injury in smaller bile ducts. Ultrastructural alterations were observed in BEC mitochondria and microvilli at 3 h with no apparent alterations in hepatocyte mitochondria or tight junctions between cells. In addition, histochemical staining of liver sections and assays of mitochondrial enzyme activities in vitro at 3 h revealed no loss of mitochondrial function in hepatocytes. These results provide strong evidence for defining DAPM as a selective bile duct toxicant.