L-arginine (L-Arg) is the substrate for nitric oxide synthase (NOS) to produce nitric oxide (NO), a signaling molecule that is key in cardiovascular physiology and pathology. In cardiac myocytes, L-Arg is incorporated from the circulation through the functioning of system-y⁺ cationic amino acid transporters. Depletion of L-Arg leads to NOS uncoupling, with O2 rather than L-Arg as terminal electron acceptor, resulting in superoxide formation. The reactive oxygen species (ROS) superoxide (O₂•^–), combined with NO, may lead to the production of the reactive nitrogen species (RNS) peroxynitrite (ONOO-), which is recognized as a major contributor to myocardial depression. In this study we aimed to determine the levels of external L-Arg that trigger ROS/RNS production in cardiac myocytes. To this goal, we used a two-step experimental design in which acutely-isolated cardiomyocytes were loaded with the dye coelenterazine that greatly increases its fluorescence quantum yield in the presence of ONOO- and O2•^–. Cells were then exposed to different concentrations of extracellular L-Arg and changes in fluorescence were followed spectrofluorometrically. It was found that below a threshold value of ~100 µM, decreasing concentrations of L-Arg progressively increased ONOO-/ O2•^–-induced fluorescence, an effect that was not mimicked by D-arginine or L-lysine and was fully blocked by the NOS inhibitor L-NAME. These results can be explained by NOS aberrant enzymatic activity and provide an estimate for the levels of circulating L-Arg below which ROS/RNS-mediated harmful effects arise in cardiac muscle.