This study determined the number of endothelial gaps in venules of the rat trachea and related these values to the amount of plasma extravasation after an inflammatory stimulus (neurogenic inflammation). From 1 to 30 min after the stimulus, vessels were fixed by vascular perfusion, and endothelial cell borders were stained with silver nitrate, which made it possible to quantify the number and distribution of endothelial gaps. It also was possible to quantify the leukocyte attachment sites, to measure the size, shape, and number of endothelial cells, and to delineate the architecture of the tracheal vasculature. Sites of increased vascular permeability were localized with Monastral blue B, india ink, or fluorescent microspheres. After the stimulus, the silver lines around endothelial cells of postcapillary venules and collecting venules were interrupted by stereotyped silver dots (diam, 1.4 +/- 0.03 microns; +/- SE), which were found by electron microscopy to be silver deposits at endothelial gaps. The dots were most abundant in the smallest postcapillary venules (diam, 7-20 microns) where Monastral blue extravasation was greatest. The number of silver dots (14.4 +/- 0.7 dots/endothelial cell) and the amount of extravasation were maximal 1 min after the stimulus. However, the dots disappeared more slowly (half-life, 3.2 min) than did the extravasation (half-life, 1.3 min). In addition to the silver dots, 64% of the sites at which leukocytes were attached to the endothelium were stained with silver. These sites were marked by silver rings (diam, 3.4 +/- 0.2 microns) and were most numerous in the largest postcapillary venules (diam, 20-40 microns). Most (95%) of the silver rings were located at intercellular junctions but usually were not sites of Monastral blue extravasation. The results indicate that endothelial gaps at intercellular junctions are focal openings, which occupy < 3% of the luminal surface and are distinct from sites of leukocyte attachment. The reduction in Monastral blue extravasation that precedes the closure of the gaps could result from a decrease in the driving force for the convective movement of the tracer or from a decrease in the conductance of the gaps, perhaps due to the accumulation of sievelike substances within the gaps.