Background: Leukocyte recruitment from the microvasculature to the site of inflammation represents a key event in the pathogenesis of inflammatory diseases. Vitronectin is a glycoprotein present in serum and extracellular matrix. Through interaction with integrins and components of the fibrinolytic system vitronectin has been implicated in cell adhesion and migration. The role of this glycoprotein in the extravasation process of leukocytes, however, remains unclear. Methods: Using in vivo microscopy on the cremaster muscle of anesthetized (ketamine, 75 mg/kg b.w. i.p., and xylazine, 25 mg/kg b.w. i.p.) wild-type and vitronectin-deficient mice, intravascular rolling and adherence as well as transmigration of leukocytes were analyzed in postcapillary venules upon intrascrotal stimulation with the C-X-C motif chemokine CXCL1/KC or lipopolysaccharide (LPS). Phenotyping of transmigrated leukocytes was performed by immunostaining of paraffin-embedded tissue sections. For statistical analysis, the ANOVA on ranks test followed by the Student-Newman-Keuls test was used. Results: Intrascrotal stimulation with CXCL1 or LPS induced a significant elevation in numbers of intravascularly firmly adherent (cells remaining stationary on the endothelium for > 30 s) and transmigrated leukocytes (> 90 % Ly-6G-positive neutrophils) as compared to PBS-treated controls. In vitronectin-deficient animals, this elevation was significantly diminished, while the number of leukocytes only temporarily adhering to microvascular endothelial cells (cells remaining stationary on the endothelium for < 30 s) was significantly increased. The average time leukocytes arrest on endothelial cells before detachment was less than 5 s in vitronectin-deficient animals. No significant differences were observed in numbers of intravascularly rolling leukocytes and in leukocyte rolling velocities among experimental groups. Conclusions: Vitronectin is critically involved in the extravasation process of neutrophils by stabilizing the adherence of these phagocytes to inflamed microvascular endothelial cells.