CD95 is a potent inducer of apoptosis. It activates the caspase cascade, but also induces ceramide (Cer) production, reportedly involving acid sphingomyelinase (aSMase) activity. A role for Cer as a second messenger for apoptosis induction was proposed, based on the finding that synthetic Cer analogues can induce cell death. We have tested whether aSMase is required for 1) apoptosis induction and 2) Cer production by CD95. For this purpose, we have used cultured Niemann-Pick disease (NPD) lymphoid cells with a defined mutation (R600H) in the aSMase protein. Despite their inherited deficiency of aSMase, we found that these cells readily undergo apoptosis upon CD95 stimulation. After retrovirus-mediated gene transfer of the aSMase cDNA, the transduced (i.e. "corrected") NPD cells showed neither increased levels of apoptosis nor altered kinetics of caspase-8 and caspase-3 activation and apoptosis induction as compared with empty vector-transduced cells. The slow sustained elevation of Cer levels in response to CD95, which we have previously documented for Jurkat T cells (Tepper, A. D., Boesen-de Cock, J. G. R., de Vries, E., Borst, J., and van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 24308-24312), was similarly found in NPD cells. Moreover, the kinetics of Cer formation remained unaffected after aSMase transduction. These results indicate that this Cer does not result from aSMase activity. We conclude that aSMase is not required for and does not facilitate CD95-mediated apoptosis and that it is not responsible for the late Cer response.