Glucocorticoid hormones (GCH) have been implicated as regulators of T-lymphocyte growth and differentiation. In particular, it has been reported that GCH can induce thymocyte apoptosis. However, the molecular mechanisms responsible for this GCH-induced death have not been clarified. In this work, the biochemical events associated with apoptosis induced by Dexamethasone (Dex), a synthetic GCH, in normal mouse thymocytes, have been analyzed. Results indicate that Dex-induced thymocyte apoptosis is attributable to an early ceramide generation caused by the activation of an acidic sphingomyelinase (aSMase). Caspase activity plays a crucial role in Dex-induced apoptosis and is downstream the aSMase activation in that inhibition of the early ceramide generation inhibits caspase activation and thymocyte death. Moreover, Dex treatment rapidly induces diacylglycerol (DAG) generation, through a protein kinase C (PKC) and G-protein-dependent phosphatidylinositol-specific phospholipase C (PI-PLC), an event which precedes and is required for aSMase activation. Indeed, PI-PLC inhibition by U73122 totally prevents Dex-induced aSMase activity, ceramide generation, and consequently, caspase activation and apoptosis. All these effects require Dex interaction with GCH receptor (GR), are countered by the GR antagonist RU486, and precede the GCH/GR-activated transcription and protein synthesis. These observations indicate that GCH activates thymocyte death through a complex signaling pathway that requires the sequential activation of different biochemical events.