The drug molsidomine is metabolized by hepatic esterases to SIN-1 that further reacts with molecular oxygen to yield nitric oxide and superoxide. Both radicals can recombine to give the harmful entity peroxynitrite. Consequently, SIN-1 is frequently used in cell culture studies as (an extracellularly operating) peroxynitrite generator. However, little is known about the nature of the reactive species produced intracellulary from SIN-1. In the present study I demonstrated that SIN-1 can easily penetrate cell membranes as exemplified with L-929 mouse fibroblasts. Intracellularly decaying SIN-1 partly generates nitric oxide as monitored by two independent methods, i.e. the fluorescent nitric oxide scavenger FNOCT-1 and the nitric oxide electrode. Moreover, reactive nitrogen-oxide species (e.g. peroxynitrite, nitrogen dioxide, dinitrogen trioxide) were intracellularly formed as detected with the scavenger 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 level of NAD(P)H was somewhat increased. This opposite behavior is conclusively explained by the capabilities of SIN-1 to simultaneously generate reactive nitrogen-oxide species (like peroxynitrite and dinitrogen trioxide) and freely diffusing nitric oxide in the intracellular milieu. These observations may lead to the conclusion that SIN-1 penetrated in vivo its target cells in order to stimulate the release of freely diffusing NO. Nitric oxide as well as RNOS decisively affect cellular metabolism, a fact that should be examined in cell culture systems by detecting the NAD(P)H level. In any case care should be taken in experimental systems to apply SIN-1 as an exclusively peroxynitrite-generating compound.