The potential role of alpha 5 beta 1 (VLA-5) in leukocyte trafficking in zymosan-induced acute peritonitis was determined. In naïve mice, approximately 98% of Gr1(high) cells (PMN) in bone marrow and circulation were alpha 5 beta 1-negative; these profiles were modestly affected by peritoneal injection of zymosan. In contrast, approximately 30% of Gr1(high) cells recruited by zymosan (24 h) to the peritoneal cavity expressed alpha 5 beta 1. With respect to F4/80(+) cells, approximately 60% of bone marrow and peripheral blood populations expressed alpha 5 beta 1, with approximately 90% positivity in resident cells of noninflamed peritoneum. Analysis of alpha 5 beta 1 expression revealed inflammation-dependent increased expression on Gr1(high) and F4/80(+) cells in bone marrow, blood, and peritoneal cavity. Blockade of alpha 5 beta 1, by an anti-alpha 5 mAb, attenuated zymosan-induced 24 h recruitment of Gr1(high) and F4/80(+) cells. At least one underlying mechanism of this action was reduction of cell adhesion and transmigration across microvascular vessels, as revealed by intravital microscopy. Confocal analyses indicated that deposition of fibronectin, the principal ligand for alpha 5 beta 1, was up-regulated significantly on and around the inflamed mesenteric microvasculature. These data suggest that the effects of alpha 5-blockade may be a result of inhibition of alpha 5 beta 1-dependent leukocyte adhesion to and migration along the fibronectin matrix. This is the first report that identifies a functional role for alpha 5 beta 1 in leukocyte trafficking during acute inflammation.