Peroxynitrite (ONOO-) is formed from superoxide (O2-) and .NO. We have previously reported that O2- does not alter endothelial cell Ca2+ signaling. To test whether .NO alters Ca2+ signaling, cells were incubated with the .NO donor, spermine NONOate. Neither spermine NONOate nor S-nitroso-N-acetyl penicillamine (SNAP) altered bradykinin-stimulated Ca2+ signaling. By contrast, 3-morpholinosydnonimine (SIN-1), which generates ONOO- by releasing O2- and .NO essentially in a simultaneous manner, significantly inhibited signaling. Initially, the inhibitory effect of 1 mM SIN-1 was selective toward agonist-stimulated influx of external Ca2+. At later time points, SIN-1 additionally depleted internal stores of releasable Ca2+. When cells were coincubated with SIN-1 plus superoxide dismutase, a technique designed to scavenge O2- and convert SIN-1 to purely an .NO-donor compound, Ca2+ signaling was identical to control. SIN-1C, the inactive metabolite of SIN-1, had no effect on [Ca2+]i. This study demonstrates that exogenously generated ONOO- modulates endothelial cell Ca2+ signaling, suggesting that ONOO- is of biological relevance to vasoregulation.