Enkephalins, endogenous opioid pentapeptides which are found in high concentration in normal chromaffin tissue, may play a role in blood pressure regulation. We therefore examined the presence and actions of enkephalins in pheochromocytoma in a rat model. Transplantable norepinephrine-rich tumors, which gave rise to significant blood pressure elevations, contained measurable immunoreactive enkephalins as determined by specific radioimmunoassays for leucine-enkephalin and methionine-enkephalin. Enkephalin immunoreactivity paralleled the enkephalin assay standard curves and was not abolished by boiling or by protease inhibitors (EDTA, PMSF). Authenticity of the immunoreactive enkephalins was confirmed by reverse-phase high pressure liquid chromatography. The amount of enkephalin immunoreactivity present initially in these tumors was greatly augmented by the prohormone activators trypsin or trypsin plus carboxypeptidase B, suggesting that most of the immunoreactive enkephalin was present in higher molecular weight precursor form. Enkephalin determinations on human pheochromocytoma and catecholamine measurements in both rat and human pheochromocytoma, demonstrated certain similarities and differences in enkephalin and catecholamine content between rat and human tumors. Total tumor enkephalins correlated (r = 0.91, p less than 0.05) with total tumor catecholamines in rat pheochromocytoma, suggesting co-regulation of synthesis of these 2 chromaffin tissue substances. Physiologic studies, in which intravenous leucine-enkephalin and the opioid antagonist nalaxone were administered to pheochromocytoma-implanted rats and sham-operated controls, failed to uncover an opioid peptide influence upon blood pressure in this animal model of pheochromocytoma.