Hydrogen peroxide (H2O2) is an inflammatory oxidant which contributes to the pathogenesis of chronic diseases such as lung injury of the respiratory tract, atherosclerosis and cancer. The mechanisms and target sites of this reactive oxidant are mainly unknown. So far there are opposing reports as to whether reactive oxidants inhibit or promote apoptosis. We activated the death pathway in primary tracheobronchial epithelial (TBE) cells with H2O2 (20-200 microM) and observed the morphological changes, DNA laddering patterns, and DNA fragmentation associated with apoptosis. Elevation of ceramide with exogenous ceramide analogs was sufficient for apoptosis induction with the same characteristics and in the same time frame. H2O2 induced rapid sphingomyelin hydrolysis to ceramide, the elevation of which paralleled the induction of apoptosis. Furthermore, H2O2 acted directly on TBE cells membrane preparations devoid of nuclei, stimulating sphingomyelin hydrolysis through a neutral Mg2+ dependent sphingomyelinase (SMase). These data suggest that the formation of ceramide from sphingomyelin in the plasma membrane is a key event in H2O2-induced apoptosis in tracheobronchial epithelial cells.