In the rat trachea, substance P causes rapid but transient plasma leakage. We sought to determine how closely the number, morphology, and size of endothelial gaps correspond to the time course of this leakage. Endothelial gaps were examined by scanning electron microscopy (EM), by transmission EM, or by light microscopy after silver nitrate staining. Substance P-induced leakage of the particulate tracer Monastral blue peaked at 1 min but decreased with a half-life of 0.3 min. The number of silver-stained gaps also peaked at 1 min then decreased significantly more slowly (half-life 1.9 min) than the leakage. Scanning EM revealed two types of endothelial gaps, designated vertical gaps and oblique slits. Vertical gaps predominated at peak leakage, whereas oblique slits became more common as the leakage diminished. Measurements of the mean diameter of vertical gaps made by light microscopy, scanning EM, and transmission EM were all in the range of 0.36-0.47 micron. Fingerlike endothelial cell processes that appeared during gap formation became shorter as the leakage diminished (mean length: 1.44 microns at 1 min compared with 1.06 microns at 3 min after substance P), suggesting a role in gap closure. We conclude that the plasma leakage occurring immediately after an inflammatory stimulus results from the rapid formation of endothelial gaps. Multiple factors, including alterations in gap morphology, gap closure, and changes in driving force, are likely to participate in the rapid decrease in the leakage.