SIN-1 is frequently used in cell culture studies as an extracellularly operating generator of peroxynitrite. However, little is known about the nature of the reactive species produced intracellulary from SIN-1. SIN-1 can easily penetrate cells as exemplified for both L-929 mouse fibroblasts and bovine aortic endothelial cells (BAECs) by utilizing capillary zone electrophoresis. In L-929 cells, SIN-1 produced nitric oxide (•NO) as monitored by the fluorescent •NO scavenger FNOCT-1 and by means of a •NO electrode, as well as reactive nitrogenoxide species (RNOS, e.g. peroxynitrite, nitrogen dioxide, dinitrogen trioxide), as detected with the fluorescent indicator DAF-2. Laser scanning microscopy revealed that in L-929 cells SIN-1-derived species initially oxidized the major fraction of the NAD(P)H within the cytosol and the nuclei, whereas the mitochondrial NAD(P)H level was somewhat increased. In marked contrast to this, in BAECs no evidence for •NO formation was found although the intracellular amount of SIN-1 was four-fold higher than in L-929 cells. In BAECs, the level of NAD(P)H was slightly decreased within the first 10 min after administration of SIN-1 in both the cytosol/nuclei and mitochondria. These observations reflect the capability of SIN-1 to generate intracellularly either almost exclusively RNOS as in BAECs, or RNOS and freely diffusing •NO as in L-929 cells. Nitric oxide as well as RNOS may decisively affect cellular metabolism as indicated by the alterations in the NAD(P)H level. Hence, care should be taken when applying SIN-1 as an exclusively peroxynitrite-generating compound in cell culture systems.