Combining single-vessel perfusion technique with confocal microscopy, this study presents a new approach that allows three-dimensional visualization and quantification of endothelial gaps under experimental conditions identical to those used to measure permeability coefficients, endothelial calcium concentration, and nitric oxide production in individually perfused intact microvessels. This approach provides an efficient means for defining the transport pathways and cellular mechanisms of increased microvascular permeability during inflammation. Platelet activating factor (PAF) was used to increase the permeability of individually perfused rat mesenteric venules. Fluorescent microspheres (FMs, 100 nm) were used as leakage markers, and confocal images were acquired at successive focal planes through the perfused microvessel. Perfusion of FMs under control conditions produced a thin, uniform layer of FMs in the vessel lumen, but in PAF-stimulated microvessels, significant amounts of FMs accumulated at endothelial junctions. Reconstructed confocal images three-dimensionally delineated the temporal and spatial development of endothelial gaps in PAF-stimulated microvessels. The FM accumulation, quantified as the total fluorescence intensity per µm² of vessel wall, was 8.4±1.8 times the control value within 10 min of PAF perfusion and declined to 5.0±0.6 and 1.4±0.2 times the control value when the FMs were applied 30 and 60 min after PAF perfusion. The changes in the magnitude of FM accumulation closely correlated with the time course of PAF-induced increases in hydraulic conductivity (Lp) indicating that the opening and closing of endothelial gaps contributed to the transient increase in Lp in PAF-stimulated microvessels. Electron microscopic evaluations confirmed PAF-induced gap formation and FM accumulation at endothelial clefts.