The heterogeneous nature of the perivascular basement membrane (composed primarily of laminin and collagen type IV) suggests the existence of an elaborate array of adhesive interactions and possibly proteolytic events in leukocyte migration through this barrier. In this context, blockade of α6 integrins (laminin receptors), neutrophil elastase (NE) or both inhibited neutrophil migration through interleukin-1β (IL-1β)-stimulated mouse cremasteric venules, as observed by intravital microscopy. Furthermore, analysis of tissues by confocal microscopy indicated a synergistic role for α6 integrins and NE in mediating neutrophil migration through the perivascular basement membrane. Using a combined in vitro and in vivo experimental approach, the findings of this study also suggest that α6 integrins and NE are mobilized from intracellular stores to the cell surface of transmigrating mouse neutrophils, although these events occur via mechanisms dependent on and independent of platelet/endothelial-cell adhesion molecule 1 (PECAM-1, CD31), respectively. Despite different regulatory mechanisms, blockade of α6 integrins or NE inhibited migration of murine neutrophils through laminin-coated filters in vitro. Collectively, the findings suggest that, whereas regulation of the expression of α6 integrins and NE occur via different adhesive mechanisms, these molecules might act in a cooperative manner in mediating neutrophil migration through venular walls, in particular the perivascular basement membrane.