We have shown previously that nitric-oxide (NO) can induce apoptosis of vascular smooth muscle cells (VSMCs) and that the NO-induced apoptosis is accompanied by an increase in arachidonic acid release via cytoplasmic Ca2+-dependent phospholipase A2 (cPLA2). We have evidence that during NO-induced apoptosis there is an increase in ceramide synthesis. The use of inhibitors of ceramide synthesis, namely, fumonisin B1 and desipramine, which block ceramide synthase and sphingomyelinase, respectively revealed that the ceramide was produced via the sphingomyelinase pathway. Inhibition of acid sphingomyelinase by desipramine was shown to inhibit NO-induced apoptosis while fumonisin B1 failed to inhibit this process. C2-ceramide could induce apoptosis in cultured VSMCs. Apoptosis in smooth muscle cells was accompanied by the increased activity of DNA fragmentation factor-40 and the secretion of cathepsin D from the cells. In this study, ceramide appears to function as a mediator of apoptosis. J. Cell. Physiol. 199: 310–315, 2004© 2003 Wiley-Liss, Inc.