In vitro and in vivo evidence supports a functional role for the integrin α₆β₁ in neutrophil migration through the perivascular basement membrane, a response that in vivo appears to be associated with platelet/endothelial cell adhesion molecule-1 (PECAM-1)-mediated up-regulation of α₆β₁ on the cell surface of transmigrating leukocytes. As the involvement of PECAM-1 in leukocyte migration is cytokine-specific, the aim of the present study was to investigate whether α₆β₁ exhibited a similar profile of stimulus specificity in this context. The cytokines interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were used to elicit neutrophil migration in two murine models of inflammation, migration through cremasteric venules, as observed by intravital microscopy, and migration into the peritoneal cavity. The role of α₆β₁ was investigated using an α₆ integrin-blocking monoclonal antibody GoH3. In both models, GoH3 significantly inhibited neutrophil transmigration induced by IL-1β but not TNF-α. This cytokine-specific role of α₆ integrin was associated with enhanced cell-surface expression of α₆β₁ on transmigrated neutrophils (as compared with blood cells) in response to IL-1β but not TNF-α. Using lipopolysaccharide as an inflammatory stimulus in the cremaster muscle model, the study also provides evidence for the involvement of α₆ integrin in leukocyte transmigration as mediated by endogenously generated IL-1β. Collectively, the findings demonstrate that α₆β₁ blockade inhibits neutrophil migration induced by exogenous and endogenous IL-1β but not TNF-α, observations that are associated with increased expression of the integrin on transmigrated leukocytes.