We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (DeltaPsim), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.