Ceramide is emerging as a putative second messenger mediating effects of extracellular agents on cell growth and differentiation (Okazaki, T., Bielawska, A., Bell, R. M., and Hannun, Y. (1990) J. Biol. Chem. 265, 15822-15831) and programed cell death (Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) Science 259, 1769-1771). In this study, the eight stereoisomers of C2-ceramide and dihydroceramide were synthesized, and their cellular activity was investigated. The four stereoisomers of C2-ceramide were active in inhibition of cell growth and induction of apoptosis with modest differences in potency. On the other hand, with C2-dihydroceramide only the threo compounds were active in these assays whereas the erythro compounds were totally inactive. Thus, of the two naturally occurring molecules, the analog of D-erythro-ceramide (with the 4-5 trans double bond) was active, whereas the analog of D-erythro-dihydroceramide was inactive. These results demonstrate the specificity of ceramide action and suggest that the introduction of the double bond is critical for imparting the biochemical and biological activity of ceramide.