The generation of ceramides by the action of acidic and/or neutral sphingomyelinases has been implicated in many forms of apoptosis. We investigated whether exposure to ceramides is sufficient to induce apoptosis in human leukemia cells and, if so, what the characteristics of this form of apoptosis might be. Treatment of the acute lymphoblastic T-cell line CEM-C7H2 with short- and medium-chain ceramide analogs (C2-, C6-, and C8-ceramide) resulted in apoptosis, whereas the inactive C2-dihydroceramide had no effect on cell survival. Induction of apoptosis was relatively slow (approximately 40% after 24 h) and required high concentrations of ceramide analogs (40-100 microM). To investigate a possible involvement of interleukin 1-beta-converting enzyme (ICE) or ICE-related proteases, we treated CEM-C7H2 sublines constitutively expressing the vaccinia virus protease inhibitor crmA with ceramide analogs. Although such cells were completely resistant to apoptosis induced by antibodies to the Apo-1/Fas surface receptor (a form of apoptosis known to be inhibitable by CrmA), they were not protected from ceramide-induced cell death. In contrast, tetracycline-regulated overexpression of Bcl-2 protected CEM-C7H2 sublines stably transfected with corresponding constructs from ceramide-induced apoptosis. Thus, in these human leukemia cells, ceramides induce a relatively slow death response that can be prevented by Bcl-2, but is independent of CrmA-inhibitable proteases. These characteristics distinguish ceramide-induced from other forms of apoptosis, such as Apo-1/Fas-induced cell death where ceramide production has been causally implicated.