Microvascular occlusion in sickle cell disease can be initiated by adhesion of sickle red blood cells (RBCs) to the endothelium. Our objective in this study was to verify the relevance in vivo of our discovery that sickle RBCs adhere abnormally to endothelial P-selectin in vitro. We used computer-assisted intravital microscopy to characterize RBC flow velocity (V_RBC) in mice. We found faster V_RBC of sickle RBCs in P-selectin knock-out and control mice than in sickle cell mice, which have increased endothelial cell P-selectin expression. Agonist peptide for murine protease-activated receptor-1 (PAR-1), which selectively activates mouse endothelial cells but not platelets, was used to assess the effects of endothelial cell P-selectin on microvascular flow. Suffusion of venules with this agonist stopped flow promptly in normal and sickle mice but not in P-selectin knock-out mice or in control mice pretreated with anti-P-selectin monoclonal antibody or unfractionated heparin (UFH). Agonist-induced slowing of flow was reversed rapidly by suffusion with UFH, provided flow had not already stopped. We conclude that endothelial cell P-selectin contributes to the microcirculatory abnormalities in sickle cell disease and that blocking P-selectin may be useful for preventing painful vasoocclusion in sickle cell disease. (Blood. 2004;104:3378-3385)