Platelet-activating factor (PAF) is a key mediator in the pathogenesis of inflammatory bowel diseases but the mechanisms of PAF-induced mucosal injury are poorly understood. In order to determine whether apoptosis and the Bcl-2-family of apoptosis regulatory gene products play a role in PAF-induced mucosal injury, we stably and conditionally over-expressed bcl-2 in rat small intestinal epithelial cells (IEC-6) under the control of a lactose-inducible promoter. Western blotting and immunohistochemistry were used to verify inducible Bcl-2 and to analyze Bcl-2 and Bax subcellular distribution. DNA fragmentation was quantified by ELISA, caspase activity was measured using fluorogenic peptide substrates and mitochondrial membrane potential was assayed by JC-1 and fluorescence digital imaging. Bcl-2 expression was highly inducible by the lactose analog isopropyl--D-thiogalactoside (IPTG) and was localized predominantly to mitochondria. In the absence of bcl-2 over-expression and following treatment with PAF, Bax translocated to mitochondria and mitochondrial membrane potential collapsed within 1 h, followed by caspase 3 activation which peaked at 6 h, with an ensuing DNA fragmentation maximizing at 18 h. Following IPTG-induction of bcl-2 expression, PAF failed to induce DNA fragmentation, caspase 3 activation, Bax translocation, or a collapse of mitochondrial membrane potential. These data are the first to show that PAF can activate the apoptotic machinery in enterocytes via a mechanism that involves Bax translocation, the collapse of mitochondrial membrane potential and that both of these events are under control by bcl-2 expression levels. A better understanding of the role of PAF and Bcl-2 family of apoptosis regulators in epithelial cell death might aid the design of better therapeutic, or preventive strategies for inflammatory bowel diseases.