Tumour Necrosis Factor-α (TNF-α) triggers cell death via apoptosis through a well defined signaling pathway activated by its p55 kDa receptor. The problem of the modulation of this signaling by intracellular second messenger is, on the contrary, a very debated and open field. In a previous paper (De Nadai et ai, 2000) we have shown that the lipidic messenger ceramide is able to increase recruitment of the TRADD protein to the p55 kDa receptor, caspase-8 activation and thus apoptosis. In the same paper we have shown that nitric oxide was able to inhibit these same features but the mechanisms through which nitric oxide and ceramide interact, however, are unclear. I have studied the contribution of ceramide-generating enzymes, i.e. the acidic and neutral sphingomyelinases (A- and N-SMase), to the apoptotic response to TNF-α, as well as their cross-talk with NO in two different cellular systems, the pro-monocytic cell line V937 and HeLa cells stably trasfected with bovine eNOS cDNA under the control of a tetracycline repressed promoter (HeLa-eNOS tet-off cell line). In U937 cells, TNF-a rapidly triggered both A- and N-SMase activity. Cells pre-incubation with the NO donor SNAP inhibited activation of both enzymes. These effects of the NO donor were mimicked by 8-Br cyclic GMP, and reverted by the guanylate cyclase inhibitor ODQ. A-SMase inhibitors reduced TNF-α triggered caspase-8 activation, while inhibition of N-SMase resulted in no effect. In HeLa cells eNOS expression was found to exert a protective effect on TNF-α induced apoptosis and, moreover, TNF-α was found to activate eNOS through a N-SMase/Pi3K/Akt dependent signalling pathway. These results suggest the existence of a regulatory loop in which NO and ceramide acts in concert in order to modulate TNF-α signalling.