The destabilization of endothelial nitric-oxide synthase (eNOS) mRNA in hypoxic endothelial cells may be important in the etiology of vascular diseases, such as pulmonary hypertension. Recently, an overlapping antisense transcript to eNOS/NOS3 was implicated in the post-transcriptional regulation of eNOS. We demonstrate here that expression of sONE, also known as eNOS antisense (NOS3AS) or autophagy 9-like 2 (APG9L2), is robustly induced by hypoxia or functional deficiency of von Hippel-Lindau protein. sONE is also up-regulated in the aortas of hypoxic rats. In hypoxic endothelial cells, sONE expression negatively correlates with eNOS expression. Blocking the hypoxic induction of sONE by RNA interference attenuates the fall in both eNOS RNA and protein. We provide evidence that the induction of sONE primarily involves transcript stabilization rather than increased transcriptional activity and is von Hippel-Lindaubut not hypoxia-inducible factor 2alpha-dependent. We also demonstrate that sONE transcripts are enriched in the nucleus of normoxic cells and that hypoxia promotes an increase in the level of cytoplasmic and polyribosome-associated, sONE mRNA. The finding that eNOS expression can be regulated by an overlapping cis-antisense transcript in a stimulus-dependent fashion provides evidence that sense/antisense interactions may play a previously unappreciated role in vascular disease pathogenesis.