Neutrophils increase volume (approximately 15%) when stimulated in suspension, but whether a similar alteration occurs in vivo during migration is unknown. We measured neutrophil volume using serial 0.5-micron sections and three-dimensional reconstruction of rabbit neutrophils migrating into inflammatory lesions in lung and abdominal wall in vivo and of human neutrophils migrating across collagen gels in vitro. An inflammatory response was induced by local instillation of C5a in vivo or generating a gradient of FMLP in vitro. In the lung, neutrophils reconstructed within the vascular space, either in arterioles (158 microns3), capillaries (128 microns3), or venules (135 microns3), were of similar volume, while those in the airspace were markedly larger (266 microns3). Neutrophils that migrated into the abdominal wall (150 microns3) were also significantly larger than those in the abdominal wall vasculature (100 microns3). Human neutrophils induced to migrate into collagen gels by FMLP were significantly larger (290 microns3) than those that did not migrate (204 microns3). We conclude from these studies that migration of rabbit neutrophils in vivo or human neutrophils in vitro is associated with a substantial increase in volume. We speculate that these findings hold promise for elucidation of the mechanisms of neutrophil migration.