Sphingomyelin signalling system has been involved in several examples of cell death through apoptosis. We have characterised the effect of exposure to the cell permeable ceramide analogue, C2-ceramide, on cultures of differentiated cerebellar granule cells. C2-ceramide was toxic to granule cells in a dose- and time-dependent way at concentrations higher than 10 μM. Ceramide exposure was accompanied by characteristic alterations of cell morphology, namely swollen cell bodies and punctuate appearance and arcuate direction of processes. The final outcome of ceramide exposure was a form of cell death largely apoptotic in nature. Hoechst stain, followed by counts of nuclei with normal appearance and size or with condensed chromatin and reduced size, revealed a large increase of the proportion of shrunken nuclei in treated cultures. In situ visualisation of fragmented DNA through the TUNEL technique, additionally marked cells undergoing apoptosis as a consequence of ceramide treatment. Accordingly, the DNA extracted from cultures exposed to C2-ceramide and subjected to agarose gel electrophoresis showed the peculiar ladder of fragmented low molecular weight DNA. Treatments with inhibitors of two caspases or of nitric oxide synthase were unable to rescue neurons exposed to ceramide, thus suggesting a neurotoxic action not primarily dependent on activation of death proteases or on nitric oxide production.