Many studies have demonstrated a biphasic effect of peroxynitrite in the myocardium, but few studies have investigated this biphasic effect on beta-adrenergic responsiveness and its dependence on contractile state. We have previously shown that high 3-morpholinosydnonimine (SIN-1) (source of peroxynitrite, 200 micromol/L) produced significant anti-adrenergic effects during maximal beta-adrenergic stimulation in cardiomyocytes. In the current study, we hypothesize that the negative effects of high SIN-1 will be greatest during high contractile states, whereas the positive effects of low SIN-1 (10 micromol/L) will predominate during low contractility. Isolated murine cardiomyocytes were field stimulated at 1 Hz, and [Ca(2+)](i) transients and shortening were recorded. After submaximal isoproterenol (ISO) (beta-adrenergic agonist, 0.01 micromol/L) stimulation, 200 micromol/L SIN-1 induced two distinct phenomena. Cardiomyocytes undergoing a large response to ISO showed a significant reduction in contractility, whereas cardiomyocytes exhibiting a modest response to ISO showed a further increase in contractility. Additionally, 10 micromol/L SIN-1 always increased contractility during low ISO stimulation, but had no effect during maximal ISO (1 micromol/L) stimulation. SIN-1 at 10 micromol/L also increased basal contractility. Interestingly, SIN-1 produced a contractile effect under only one condition in phospholamban-knockout cardiomyocytes, providing a potential mechanism for the biphasic effect of peroxynitrite. These results provide clear evidence for a biphasic effect of peroxynitrite, with high peroxynitrite modulating high levels of beta-adrenergic responsiveness and low peroxynitrite regulating basal function and low levels of beta-adrenergic stimulation.