Background and Objectives The mechanism of glucocorticoid -induced apoptosis is not fully understood and early predictive assays based on apoptotic markers for clinical outcome in acute lymphoblastic leukemia (ALL) are scarce. The aim of this study was to characterize the involvement of Bcl-2 family members and caspase activation in dexamethasone(Dex)induced apoptosis in ALL. Design and Methods Primary childhood ALL samples, the pre-B ALL cell line RS(4;11), and the T-ALL cell line CCRF-CEM were used. The involvement of Bcl-2 family members was evaluated by flow cytometry, immunocytochemistry, and western and northern blotting. Apoptosis was analyzed by annexin V and TMRE staining. Caspase activity was evaluated by a fluorometric assay. Results Dex induced significant down-regulation of the anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, differential activation of the pro-apoptotic Bak and Bax, loss of Delta psi m and cytochrome c release. Dex-induced apoptosis also involved early activation of caspases 2 and -3. Inhibition of caspase activity did not, however, protect against Dex-induced Bak/Bax activation, loss of Delta psi m or cell death. In 12 primary ALL samples Dex-induced apoptosis was associated with activation of Bax (p=0.045) and clown-regulation of Bcl-2 (p=0.016) and/or Bcl-xL (p=0.004). Furthermore, ex vivo Dex-sensitivity was associated with an early treatment response to polychemotherapy (p=0.026). Interpretation and Conclusions The differential regulation of pro- and anti-apoptotic Bcl-2 family members appears to be a key event in the execution of Dex-induced apoptosis in ALL cell lines, and also indicates a role for these proteins in primary ALL cells.