We studied the inhibition of tumour necrosis factor alpha (TNFalpha)- and camptothecin-induced apoptosis by Bcl-2 and Bcl-xL as they relate to the ceramide pathway. Expression of either Bcl-2 or Bcl-xL provided significant protection from the apoptotic effects of TNFalpha or camptothecin. In contrast to Bcl-2, Bcl-xL overexpression did not protect cells from ceramide-induced apoptosis. On the other hand, Bcl-xL prevented the accumulation of endogenous ceramide in response to TNFalpha or camptothecin, whereas Bcl-2 showed little effect on ceramide formation. Moreover, Bcl-xL, but not Bcl-2, totally inhibited a caspase-8-like activity in cell lysates stimulated with TNFalpha. These results identify a different mechanism of action for Bcl-xL compared with Bcl-2 and they demonstrate that Bcl-xL targets a point upstream of ceramide generation, whereas Bcl-2 functions downstream of ceramide in the TNFalpha- and camptothecin-activated pathways of apoptosis.