In vitro binding of (3-[125I]Tyr)-endothelin-1 ([125I]ET-1) and (3-[125I]Tyr)-big ET-1(1-38) ([125I]big ET-1) to plasma proteins of healthy humans, cardiac patients and normotensive and hypertensive rats was investigated by equilibrium dialysis. Binding of both tracers was similar in plasma from healthy humans, patients with congestive heart failure, and following myocardial infarction (approximately 60%), and marginally higher in rat plasmas (approximately 70%). Binding of [125I]ET-1 to human plasma could be explained by binding to human serum albumin. Endogenous plasma ET-1 levels were approximately 9 pg/ml in healthy humans, and approximately 12-16 pg/ml in cardiac patients; big ET-1 concentrations were approximately two- to threefold higher. ET-1 bound to plasma protein was partly lost in column extraction. In rat isolated perfused hearts, the coronary dilator and constrictor potency of exogenous free and albumin-bound ET-1 was similar, whereas the kinetics of endogenous ET-1 was impeded by tight binding to ET receptors. The data indicate that binding of ET-1 to plasma proteins is without effect on peptide vasoactivity, but binding to tissue receptors greatly impedes its tissue kinetics.