The objective of this study was to systematically assess the molecular mechanisms and kinetics of histamine-induced leukocyte rolling in rat mesenteric venules using intravital microscopy. A complicating factor in these studies is surgical preparation-induced leukocyte rolling (spontaneous rolling), which leads to a lack of effect of histamine on this parameter. Therefore, we identified the source of the surgery-induced leukocyte rolling (partial mast cell degranulation) and established that pretreatment of animals with sodium cromoglycate (connective tissue mast cell stabilizer) inhibited spontaneous leukocyte rolling. Superfusion of the mast cell-stabilized rat mesentery with histamine caused a profound increase in leukocyte rolling which persisted for the entire hour of experimentation. Diphenhydramine (H1-receptor antagonist) but not cimetidine (H2-receptor antagonist) prevented the rise in histamine-induced leukocyte rolling. An anti-P-selectin Ab but not an anti-CD18 Ab reversed the histamine-induced leukocyte rolling in a dose-dependent fashion. In this model of low base line rolling, exposure of the mesentery to the chemotactic agent platelet-activating factor did not induce leukocyte rolling or adhesion. However, co-administration of histamine with platelet-activating factor did indeed promote leukocyte adhesion suggesting that the presence of at least one effector of P-selectin is a minimal requirement for chemotactically-stimulated leukocytes to adhere to postcapillary venules. This study demonstrates for the first time that histamine induces leukocyte rolling via a P-selectin-dependent mechanism in vivo. This is a prolonged, H1 receptor-mediated event that may contribute significantly to the early phase of inflammation.