Platelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear. The present results demonstrate that the ability of alpha(6) integrins to mediate neutrophil migration through the PBM is PECAM-1 dependent, a response associated with PECAM-1-mediated increased expression of alpha(6)beta(1) on transmigrating neutrophils in vivo. An anti-alpha(6) integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1beta-stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy. In PECAM-1-deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1beta (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses. FACS((R)) analysis of neutrophils demonstrated increased expression of alpha(6)beta(1) on transmigrated peritoneal neutrophils, as compared with blood neutrophils, in wild-type but not KO mice even though neutrophils from both strains of mice exhibited comparable levels of intracellular expression of alpha(6) as observed by immunofluorescent staining and confocal microscopy. Furthermore, mice deficient in either leukocyte or endothelial cell PECAM-1, as developed by bone marrow transplantation, demonstrated a similar level of reduced neutrophil transmigration and expression of alpha(6)beta(1) on transmigrated neutrophils as that detected in KO mice. The results demonstrate a role for PECAM-1 homophilic interaction in neutrophil transmigration and increased expression of alpha(6)beta(1) on the cell surface of transmigrated neutrophils in vivo, a response that could contribute to the mechanism of PECAM-1-mediated neutrophil migration through the PBM.