The site of neutrophil interaction with the vasculature during acute lung inflammation is controversial, but has been suggested to occur in the alveolar capillaries, in contrast with its location in postcapillary venules in nonpulmonary tissues. We studied the kinetics of neutrophil accumulation and the site of neutrophil-vascular interaction in the lung by examining directly the behavior of fluorescein isothiocyanate-labeled canine neutrophils utilizing in vivo fluorescence videomicroscopy through a window inserted into the chest wall of anesthetized dogs. The administration of fragments of the fifth component of complement (C5f) into either the airway or pulmonary artery resulted in neutrophil sequestration almost exclusively in pulmonary capillaries. Kinetically, there was a shift in the distribution of neutrophil transit times resulting in a marked prolongation of median transit time. This response occurred within seconds after intravascular C5f and within 5 minutes after airway C5f and was maintained for at least 30 minutes. Ultrastructural studies after airway C5f showed neutrophils in various stages of migration through the alveolar-capillary membrane and more than 90% of these neutrophils were seen to migrate from capillary rather than from venular sites. These data indicate that pulmonary inflammation differs from inflammation in other vascular beds primarily in the site of neutrophil localization and migration. This fundamental difference in the inflammatory response may serve to localize the inflammatory response to the alveolus, and (since cells were retained singly), indicates the inability of leukoaggregation adequately to explain the findings. Leukocyte accumulation in the lung may thus occur through alterations in the balance between delivery of neutrophils to the lung and the transit time of these cells across the capillary bed.