The production of peroxynitrite during 3-morpholinosydnonimine (SIN-1) decomposition can be continuously monitored, with a sensitivity ≤ 0.1 μM, from the kinetics of NADH fluorescence quenching in phosphate buffers, as well as in buffers commonly used with cell cultures, like Locke's buffer or Dulbecco's modified Eagle's medium (DMEM-F12). The half-time for peroxynitrite production during SIN-1 decomposition ranged from 14–18 min in DMEM-F12 (plus and minus phenol red) to 21.5 min in Locke's buffer and 26 min in DMEM-F12 supplemented with apotransferrin (0.1 mg/mL). The concentration of peroxynitrite reached a peak that was linearly dependent upon SIN-1 concentration, and that for 100 μM SIN-1 amounted to 1.4 ± 0.2 μM in Locke's buffer, 3.2–3.6 μM in DMEM-F12 (plus and minus phenol red) and 1.8 μM in DMEM-F12 supplemented with apotransferrin. Thus, the maximum concentration of peroxynitrite ranged from 1.2 to 3.6% of added SIN-1. NADH was found to be less sensitive than dihydrorhodamine 123 and 2′,7′-dichlorodihydrofluorescein diacetate to oxidation by H₂O₂, which is produced during SIN-1 decomposition in common buffers. It is shown that peroxynitrite concentration can be controlled (±5%) during predetermined times by using sequential SIN-1 pulses, to simulate chronic exposure of cells or subcellular components to peroxynitrite.