The Reaper (Rpr) gene encodes a 65-amino acid protein that induces apoptosis in Drosophila by an unknown mechanism. A previous study reported that Rpr expression induced generation of the lipid second messenger ceramide and through use of the peptide caspase inhibitor N-benzyloxycarbonyl-VAD- fluoromethylketone (zVAD.fmk) ordered ceramide generation downstream of caspases in SL2 cells (Pronk, G. J., Ramer, K., Amiri, P., and Williams, L. T. (1996) Science 271, 808-810). The present study re-evaluates these events in SL2 cells transfected with cDNA for Rpr, with or without the baculovirus caspase inhibitor p35, under the control of the metallothionein promoter. Following copper addition, Rpr protein was detected at 1.5 h and maximal at 2.5 h. Ceramide generation and caspase activation occurred nearly simultaneously, each detectable at 2-2.5 h and maximal at 6 h. Ceramide levels increased from a base line of 5 pmol/nmol lipid phosphorus to a maximum of 10 pmol/nmol lipid phosphorus. Identical increases in ceramide were detected using the enzymatic 1,2-diacylglycerol kinase assay or the non- enzymatic o-phthalaldehyde derivatization high pressure liquid chromatography assay. In contrast, diacylglycerol levels were not increased by Rpr expression. Apoptosis, first detected at 4 h, was maximal at 16 h. Co- expression of p35 did not affect Rpr-induced ceramide generation, whereas caspase activation and apoptosis were abolished. In contrast, zVAD.fmk inhibited ceramide generation and apoptosis. These data show that Rpr- induced ceramide generation is upstream or independent of p35-inhibitable caspases and demonstrate differences in the actions of peptide and p35 caspase inhibitors.