The liver of adult rats with cholestasis secondary to bile duct resection has been shown to express the proenkephalin gene and, by immunohistochemical stains, to contain met-enkephalin. To further study hepatic opioids in cholestasis, concentrations of proenkephalin-derived endogenous opioids were measured in a rat model of cholestasis by the use of radioimmunoassays. The specificity of the immunoreactivity detected by the assays was confirmed by high performance liquid chromatography (HPLC). In adult male rats with cholestasis due to BDR, the concentrations of three proenkephalin-derived opioid peptides were increased. Specifically, the mean hepatic concentrations of met-enkephalin, Met-Enk-Arg6-Phe7 and leu-enkephalin were 2.5 (p < 0.005), 2.1 (p < 0.005) and 2.5 (p < 0.01) fold higher than the corresponding mean for controls. These findings provide further independent evidence that opioid peptides accumulate in the liver in a model of cholestasis and are consistent with de novo synthesis of opioid peptides occurring in the cholestatic liver. This phenomenon may have relevance to the altered function of the opioid system in cholestasis and to the role of the liver as a neuroendocrine organ.