Dependence of pyruvate's positive inotropic effect on energetic substrate availability and potential role of its mitochondrial uptake were investigated. Pyruvate (3, 10 and 15 mM) was added to rabbit right ventricular papillary muscles (protocol I; n = 10) and human right auricular trabeculae (protocol II; n = 6), using glucose as energetic substrate. In protocols III & IV (rabbit papillary muscles; n = 8 and n = 10, respectively) pyruvate's mitochondrial uptake was inhibited by alpha-cyano-4-hydroxycinnamate (0.5 mM) with octanoate as energetic substrate at 5 and 0.2 mM, respectively. In 8 additional rabbit papillary muscles, effects of L-alanine (10, 20 and 50 mM) were tested. In protocols I&II, pyruvate had a dose-dependent positive inotropic effect that was maximal at 10 mM, increasing in rabbit myocardium: 45.0+/-9.4% active tension, 20.5+/-7.4% peak rate of tension rise, 32.5+/-8.6% peak isotonic shortening, 31.2+/-11.7% peak rate of lengthening, 27.8+/-3.2% twitch duration. In protocol III (5 mM octanoate), pyruvate's positive inotropic effect was still present and even enhanced, while in protocol IV (0.2 mM octanoate) it was decreased and not observed with 3 mM of pyruvate. We conclude that, in rabbit papillary muscles, the positive inotropic effect of pyruvate is modulated by the availability of metabolic substrates and presumably does not depend on its mitochondrial uptake.