In the initial phase of an inflammatory response, leukocytes marginate and roll along the endothelial surface as a result of adhesive interactions between molecules on the endothelial cells and leukocytes. To evaluate the role of the 3 selectins (E, L, and P) in leukocyte rolling and emigration, a null mutation for L-selectin was introduced into previously described embryonic stem cells with null mutations in the genes for both E-selectin and P-selectin (E/P double mutants) to produce triple-selectin-null mice (E-selectin, L-selectin, and P-selectin [E/L/P] triple mutants). Triple-selectin homozygous mutant mice are viable and fertile and only rarely develop the severe mucocutaneous infections or pulmonary inflammation characteristic of E/P double-mutant mice. Surface expression of L-selectin was undetectable in triple-mutant mice on fluorescence-activated cell-sorter analysis of peripheral neutrophils. Pathological studies revealed moderate cervical lymphadenopathy and lymphoplasmacytic infiltrate, but these were less extensive than in E/P double-mutant mice. Neutrophil emigration during thioglycolate-induced peritonitis was significantly reduced at 4, 8, and 24 hours (35%, 65%, and 46% of wild-type values, respectively). Intravital microscopy of the cremaster muscle revealed almost no rolling at times up to 6 hours after exteriorization, with or without addition of tumor necrosis factor alpha. The small amount of residual rolling was dependent on alpha(4)-integrin. The occurrence of skin and pulmonary disease in E/P double-mutant mice but not E/L/P triple-mutant mice suggests that deficiency of L-selectin alters the inflammatory response in E/P mutants. (Blood. 2001;98:727-735)