The novel lipid second messenger, ceramide, induced apoptosis in PC12 cells as determined morphologically by nuclear appearance and internucleosomal DNA fragmentation. Apoptosis was induced by exogenous C₂-ceramide in a dose- and time-dependent manner. Natural ceramide and C₆-ceramide had a similar effect. This response was specific since the structural analog C₂-dihydroceramide and other related lipids failed to initiate apoptosis. The apoptotic effect of ceramide also depends critically on cell plating density. Furthermore, the peptide inhibitor of interleukin-1β converting enzyme (ICE)-like proteases, Z-VAD.FMK, completely prevented the nuclear changes induced by ceramide, implicating the involvement of ICE-like protease activation in ceramide-induced apoptosis in PC12 cells.