In diabetic retinopathy, endothelial cell apoptosis is paradoxically increased despite upregulation of the potent pro-survival factor VEGF. We tested the hypothesis that elevated glucose levels disrupt VEGF's pro-survival function via peroxynitrite-mediated alteration of the Akt-1/p38 MAP kinase signaling pathway by studies of retinal endothelial cells in vitro. High glucose or exogenous peroxynitrite caused significant increases in apoptosis in the presence or absence of VEGF. Treatment with a peroxynitrite decomposition catalyst blocked these effects, implying a causal role of peroxynitrite. Peroxynitrite or high glucose treatment also increased phosphorylation of p38 MAP kinase, whereas phosphorylation of Akt-1 was significantly decreased in basal or VEGF-stimulated conditions. High glucose- or peroxynitrite-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI 3-kinase that blocked PI 3-kinase and Akt-1 kinase activity. Inhibiting peroxynitrite formation or blocking tyrosine nitration of p85 restored the activity of PI 3-kinase and Akt-1 kinase, blocked phosphorylation of p38 MAP kinase and normalized pro-survival function. Transfecting the cells with constitutively active Akt-1 or inhibiting activity of p38 MAP kinase completely masked the pro-apoptotic effects of high glucose and exogenous peroxynitrite, suggesting an interaction between the Akt-1 and p38 MAP kinase pathways. In conclusion, high glucose treatment blocks the pro-survival effect of VEGF and causes accelerated endothelial cell apoptosis via the action of peroxynitrite in causing tyrosine nitration of PI 3-kinase, inhibiting activity of Akt-1 kinase and increasing the activity of p38 MAP kinase.