Previous in vivo studies indicate that prolonged exposure to NO+HYP causes significant lung injury in newborn piglets. Peroxynitrite-mediated oxidant damage has been implicated in the pathogenesis of this injury. However, little is known about the in vivo mechanisms of cell injury or the mode of subsequent cell death. Our previous in vitro studies in human alveolar epithelial (A549) cells demonstrated that NO+HYP directly induced acute and synergistic cytotoxicity, but the mode of cell death was found to be nonapoptotic. To determine if NO+HYP results in apoptosis in vivo, we studied 1-3 day old piglets, mechanically ventilated for 48h with 100% O2, 100 PPM NO + 21% O2, 5 PPM NO + 100% O2, 100 PPM NO + 90% O2 or unventilated control animals(n=2/group). Animals were sacrificed, lungs removed and instillation-fixed in buffered formalin and embedded in paraffin. TUNEL (TdT dUTP Nick End Labeling) assays were performed on sections of lung tissue (right lower lobe) and the number of apoptotic (TUNEL+) nuclei/field were quantified. Unventilated animals had 3.5±0.5 (mean ± SE) apoptotic nuclei/field while animals ventilated with 100 PPM NO + 21% O2 had 7.0±2, 100% O2 had 27±3, 5 PPM NO + 100% O2 had 31±4, and 100 PPM NO+ 90% O2 had 68±9 (p< 0.05, ANOVA). Preliminary data suggest that combined therapy with NO+HYP results in increased apoptosis in the lung which may correlate with previously documented pulmonary injury.