Purpose: The nitric oxide pathway requires phosphorylation of eNOS to liberate NO. We correlated mRNA/protein levels of VEGF164, and phosphorylated Serine1179 of eNOS during ROP vasoproliferation. Methods: Animals were exposed to oxygen (50/10% every 24-hours) for 14 post-natal (PN) days. Pups were continuously exposed to 20.9% ambient oxygen from PN15 and enucleated on PN days 15, 18, and 20. ADPase stained retinas were analyzed for degree of avascularity/total retina area. Western-blotting quantified the phosphorylation of eNOS at Ser1179, the ratio of total eNOS to ERK2. Immunoreactivity of VEGF and of eNOS serine1177 determined tissue protein expression. Results: Percent retinal avascularity was reduced (p<0.05) from 39.69% to 28.79%, to 18.53%, for PN days 15, 18, and 20, respectively, with elevations of VEGF mRNA, and eNOS. Phosphorylation of eNOS at Ser1179 was greater at PN20 than PN15 and control. Immunoreactivity of VEGF and eNOS increased. Conclusion: Findings provide insight into mechanisms involving specific interrelationships between VEGF and phosphorylated eNOS in the vasoproliferation of ROP.