It has not been determined whether L-selectin-mediated rolling can promote leukocyte adhesion in vivo independent of P- and E-selectin. We used intravital microscopy of E- and P-selectin double-mutant mice (E-/P-) stimulated with tumor necrosis factor-α for 6-8 h to investigate the importance of L-selectin-dependent rolling in cremaster muscle venules. Rolling leukocyte flux in E-/P- mice was 9 ± 2 cells/min compared with 77 ± 17 cells/min in wild-type (WT) mice. Pretreatment with the L-selectin monoclonal antibody MEL-14 significantly reduced rolling in both E-/P(by 89%) and WT mice (by 79%). L-selectin-dependent rolling in E-/P- mice resulted in leukocyte adhesion comparable to that seen in WT mice. MEL-14 pretreatment of E-/P- mice reduced leukocyte adhesion by 50%. The majority (~80%) of intravascular leukocytes in both WT and E-/P- mice were neutrophils. We conclude that L-selectin can mediate rolling that results in sufficient leukocyte recruitment to account for the robust inflammatory response seen in E-/P- mice at later times.