Nitric oxide (NO) is a potent vasodilator being used clinically to treat pulmonary hypertension in infants and children. We have previously demonstrated that NO and HYP have synergistic cytotoxic effects on both cultured human alveolar epithelial cells (A549) and human microvascular endothelial lung cells. To better understand the basis of this cytotoxicity, we studied the effects of combined NO and HYP on HeLa 80 cells, a substrain of HeLa cells capable of growing normally in 80% oxygen. Cultured HeLa 80 cells were grown under 4 separate experimental conditions:1) room air (RA), 2) 80% oxygen, 3) NO donor (0.5mM NONOate - ethanolamine, 2,2'-hydroxynitrosohydrazono bis-) and RA, 4) combined 0.5mM NONOate and 80% oxygen. Live cells were counted daily (using Trypan blue) in each of the above groups for six consecutive days, and culture media and gasses were replenished each day. Cells continued to grow normally in RA or 80% oxygen. Cells grew more slowly with the NO donor and RA. In sharp contrast, cells treated with the combination of the NO donor and 80% oxygen began to die on day 3, with approximately 75% of the total cells having died by day 6. To study the mode of cell death, cells were grown on cover slips and divided into the 4 experimental conditions as described above. The cells were then stained with DAPI (4',6-diamidine-2-phenylindole -dihydrochloride), a DNA binding dye and viewed under a fluorescent microscope. Significant numbers of cells were found to be apoptotic when treated with the NO donor + 80% oxygen. These data indicate that HeLa 80 cells, which are normally resistant to the effects of HYP, die rapidly when exposed to combined NO and HYP. This suggests that NO and HYP are synergistically cytotoxic and the most significant mode of cell death is apoptosis. This is different from typical hyperoxia-sensitive cells, which do not undergo apoptosis in combined NO + HYP.