We tested the hypothesis that VEGF regulates endothelial hyperpermeability to macromolecules by activating the ERK-1/2 MAPK pathway. We also tested whether PKC and nitric oxide (NO) mediate VEGF-induced increases in permeability via the ERK-1/2 pathway. FITC-Dextran 70 flux across human umbilical vein endothelial cell monolayers served as an index of permeability, whereas Western blots assessed the phosphorylation of ERK-1/2. VEGF-induced hyperpermeability was inhibited by antisense DNA oligonucleotides directed against ERK-1/2 and by blockade of MEK and Raf-1 activities (20 μM PD-98059 and 5 μM GW-5074). These blocking agents also reduced ERK-1/2 phosphorylation. The PKC inhibitor bisindolylmaleimide I (10 μM) blocked both VEGF-induced ERK-1/2 activation and hyperpermeability. The NO synthase (NOS) inhibitor N G-nitro-l-arginine methyl ester (200 μM) and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidiazoline-1-oxyl-3-oxide (100 μM) abolished VEGF-induced hyperpermeability but did not block ERK-1/2 phosphorylation. These observations demonstrate VEGF-induced hyperpermeability involves activation of PKC and NOS as well as Raf-1, MEK, and ERK-1/2. Furthermore, our data suggest that ERK-1/2 and NOS are elements of different signaling pathways in VEGF-induced hyperpermeability.