The characteristics of endothelin-1 (ET-1) and endothelin-3 (ET-3) binding, and their relationship to second messenger formation in vitro and vascular effects in vivo were studied in the pig. Specific high-affinity binding sites for [125I]ET-1 and [125I]-ET-3 with extremely slow dissociation rates were demonstrated in membrane preparations from the spleen, lung, kidney and spinal cord. Displacement studies showed that receptor populations with much higher affinity for ET-1 than for ET-3 (ETA type) were present in the spleen and renal arteries, while in the whole kidney and spinal cord, receptor populations with similar affinity for ET-1 and ET-3 were found (ETB type). In the lung both receptor subtypes may be present. The precursor forms big ET-1 and big ET-3 were poor ligands although big ET-1 was more active on the ETA site than big ET-3 on the ETB site. Scatchard analysis revealed linear plots in all tissues studied. Both ET-1 and ET-3 increased formation of inositol phosphates in the lung, while ET-1 but not ET-3 was effective in the spleen. Neither ET-1 nor ET-3 were observed to influence basal or stimulated cyclic AMP formation in lung or spleen. ET-1 caused a much more potent and long-lasting increase in splenic and renal vascular resistance in vivo than did ET-3. On the other hand, ET-1 and ET-3 decreased vascular resistance with almost equal potency in the bronchial circulation.