Ceramide, a product of sphingomyelin metabolism, is a novel lipid second messenger that mediates diverse cellular functions. The present study demonstrates the activation of caspase-3/CPP-32beta, during apoptosis induced by cell permeable exogenous ceramides, in AK-5 tumor, a spontaneously regressing rat histiocytoma. The apoptotic events were suppressed by the caspase-3 specific tetrapeptide inhibitor DEVD-CHO but not by the caspase-1 inhibitor YVAD-CMK. In cells overexpressing Bcl-2, a significant decrease in cell death was observed after exogenous addition of ceramides. Furthermore the processing of caspase-3 to its active form upon apoptotic stimulus, and the subsequent cleavage of the substrate PARP, suggested a central role for caspase-3 in the ceramide mediated apoptosis in AK-5 tumor cells.