Endothelial nitric oxide synthase (eNOS) is thought to regulate microvascular permeability via NO production. We tested the hypotheses that expression of eNOS and eNOS endocytosis by caveolae are fundamental for appropriate signaling mechanisms in inflammatory endothelial permeability to macromolecules. We used bovine coronary postcapillary venular endothelial cells (CVEC) because these cells are derived from the microvascular segment responsible for transport of macromolecules in inflammation. We stimulated CVEC with platelet-activating factor (PAF) at 100 nM and measured eNOS phosphorylation, NO production and CVEC monolayer permeability to FITC-dextran-70. PAF translocated eNOS from plasma membrane to cytosol, induced changes in the phosphorylation state of the enzyme and increased NO production from 4.3 ± 3.8 to 467 ± 22.6 nM. PAF elevated CVEC monolayer permeability to FITC-dextran-70 from 3.4 ± 0.3 x 10^-6 cm/s to 8.5 ± 0.4 x 10^-6 cm/s. Depletion of endogenous eNOS with siRNA abolished PAF-induced hyperpermeability demonstrating that expression of eNOS is required for inflammatory hyperpermeability responses. Inhibition of the caveolar internalization by blocking caveolar scission using transfection of dyn2K44A (dynamin dominant negative mutant) inhibited PAF-induced hyperpermeability to FITC-dextran-70. We interpret these data as evidence that 1) eNOS is required for hyperpermeability to macromolecules, and 2) internalization of eNOS via caveolae is an important mechanism in the regulation of endothelial permeability. We advance the novel concept that eNOS internalization to cytosol is a signaling mechanism for onset of microvascular hyperpermeability in inflammation.