L-Methionine is an essential amino acid that has been reported to have a potent positive inotropic effect on the mammalian myocardium. We studied the mechanisms of the inotropic effect in ventricular myocardium from the rabbit. In the isolated coronary-perfused whole heart, L-methionine in a millimolar range exerted concentration-dependent positive inotropic effects on the isovolumic left ventricle, which were associated with negative lusitropic effects (prolonged time course of relaxation). The chronotropic state and the coronary perfusion pressure were not affected. These complex effects on the isolated whole heart were not blocked by pretreatment with (mumol/L) propranolol 1, prazosin 1, carbachol 3, staurosporine 1, or [Ser1,Ile8]angiotensin II 0.1. To further study the subcellular mechanisms, isolated ventricular papillary muscles from the same species were loaded with a bioluminescent indicator, aequorin, to monitor [Ca2+]i. In the presence of 3 mmol/L L-methionine, the isometric tension showed a similar combination of the positive inotropic and negative lusitropic effects as observed in the whole heart. In contrast, the simultaneously recorded intracellular Ca2+ signals did not increase in amplitude but instead decreased. The [Ca2+]i-tension relation shifted to the left compared with that obtained in response to [Ca2+]o. In saponin (250 micrograms/mL)-treated skinned preparations, 3 mmol/L L-methionine also shifted the force-pCa curve to the left by 0.16 pCa units. This is the first demonstration that an essential amino acid directly acts on the myofilaments and modulates their responsiveness to Ca2+, thereby producing a positive inotropic effect.