Activation of acid and neutral sphingomyelinases, and the ensuing generation of ceramide, contributes to the biological effects of tumour necrosis factor-alpha (TNF-alpha), one of which is apoptosis. While the mechanisms of activation of sphingomyelinases by the cytokine are being unravelled, less is known about regulation of their activity. Nitric oxide has previously been shown to exert a cyclic GMP-dependent inhibition of early apoptotic events triggered by TNF-alpha in the U937 monocytic cell line. We therefore investigated whether inhibition of sphingomyelinases by nitric oxide plays a role in regulating such early events. We found that activation of both acid and neutral sphingomyelinases, triggered in the first minutes after U937 cell stimulation with TNF-alpha, is regulated in an inhibitory fashion by nitric oxide, working through generation of cyclic GMP and activation of protein kinase G. Using a range of inhibitors selective for either sphingomyelinase we found that the acid sphingomyelinase contributes to activation of the initiator caspase-8 and early DNA fragmentation and that inhibition of the acid enzyme by nitric oxide accounts for cyclic GMP-dependent early protection from apoptosis. We also found that the protective effect by both cGMP and acid sphingomyelinase inhibitors progressively disappeared at later stages of the apoptotic process. Inhibition of sphingomyelinases represents a novel action of nitric oxide, which might be of physiological relevance in regulating initial phases of apoptosis as well as other biological actions of ceramide.