The present study was designed to characterize endothelium-derived relaxing factor (EDRF) and nitric oxide (NO) by employing both biological and chemical methods. EDRF was released by the calcium ionophore A23187 from cultured bovine pulmonary artery endothelium (BPAE) grown on microcarrier beads and then superfused in a cell column. The maximum relaxations induced by EDRF (83%) or NO (79%) on phenylephrine (PE)-contracted rabbit aorta were similar. In contrast, EDRF was only half as potent as NO in relaxing the KCl-contracted rabbit aorta. EDRF induced a concentration-dependent relaxation of both PE-contracted rabbit aorta and histamine-contracted guinea pig aorta that was accompanied by a marked elevation in cyclic GMP levels. However, EDRF was vascular selective and did not relax or increase cyclic GMP levels of the histamine-contracted taenia coli of either species. NO was not vascular selective and relaxed both aorta and taenia coli and also markedly increased cyclic GMP levels in each. NO also relaxed dog femoral artery and gastrointestinal smooth muscle preparation of the lower esophageal sphincter, whereas EDRF only relaxed the femoral artery. Experiments were also performed describing the actions of a series of different resins: anion exchange resins (NH2/NH, AG-1), cation exchange resin (-COOH), reversed phase resin (C18) and hemoglobin-agarose on EDRF- or NO-induced relaxation. NH2/NH, AG-1 and hemoglobin-agarose resins inhibited EDRF-induced relaxation, but -COOH and C18 did not. The inhibition was dependent on the amount of resin employed. NO-induced relaxation was blocked only by hemoglobin-agarose but by none of the other resins.(ABSTRACT TRUNCATED AT 250 WORDS)