The signaling events leading to the activation of integrins and firm arrest of rolling neutrophils in inflamed venules have yet to be elucidated. In vitro assays suggest that both E-selectin and chemokines can trigger arrest of rolling neutrophils, but E-selectin^−/− mice have normal levels of adherent neutrophils in inflamed venules. To test whether chemokine-induced neutrophil arrest in vivo can be unmasked by blocking E-selectin, we investigated neutrophil adhesion in inflamed cremaster muscle venules in tumor necrosis factor (TNF)-α–treated CXCR2^−/− or wild-type (WT) mice injected with E-selectin blocking monoclonal antibody (mAb) 9A9. To block chemokine receptor signaling, we investigated E-selectin^−/− or WT mice treated with pertussis toxin (PTx) intravenously. Neutrophil adhesion was unchanged in CXCR2^−/−, E-selectin^−/−, PTx-treated WT, or mAb 9A9–treated WT mice. However, TNF-α–induced neutrophil adhesion was almost completely abrogated in E-selectin^−/− mice treated with PTx and significantly reduced in CXCR2^−/− mice treated with the E-selectin blocking mAb. In thioglycollate-induced peritonitis, PTx treatment blocked neutrophil recruitment into the peritoneum of E-selectin^−/− mice, but had only a partial effect in WT animals. These data show that E-selectin– and chemokine-mediated arrest mechanisms are overlapping in this model and identify CXCR2 as an important neutrophil arrest chemokine in vivo.