Rolling on the venular endothelium is a critical step in the recruitment of leukocytes during the inflammatory response. P-selectin is a key mediator of leukocyte rolling, which is an early event in the inflammatory cascade; this rolling is likely to be directly regulated by both local fluid shear forces and P-selectin site densities in the microvasculature. However, neither the spatial pattern of P-selectin expression in postcapillary venules nor the effect of local expression patterns on rolling behavior in intact functional venules is known. We investigated the influence of local shear forces and the spatial distribution of endothelial P-selectin in intact blood perfused post capillary venules in anesthetized mice using intravital confocal microscopy, high temporal resolution particle tracking, and immunofluorescent labeling. We demonstrated a shear-dependent increase in average leukocyte rolling velocity that was attributable to a shear-dependent increase in the occurrence of transient leukocyte detachments from the endothelial surface: translational velocity during leukocyte contact with the vessel wall remained constant. P-selectin expression was not different in venules with characteristically different shear rates or diameters but varied significantly within individual venules. In postcapillary venules, regions of high P-selectin expression correlated with regions of slow leukocyte rolling. Thus the characteristically variable leukocyte rolling in vivo is a function of the spatial heterogeneity in P-selectin expression. The study shows how the local hydrodynamic forces and the nonuniform pattern of P-selectin expression affect the behavior of interacting leukocytes, providing direct evidence for the local variation of adhesion molecule expression as a mechanism for the regulation of leukocyte recruitment.