Recruitment of circulating leukocytes to the site of infection is regulated by chemokines sequestered on e.g. heparan sulfate (HS) on endothelial cells. A new step in the leukocyte recruitment cascade was recently described, namely intraluminal crawling of neutrophils to distant, optimal sites for emigration. The aim of this study was to in vivo determine if neutrophil crawling is influenced by a chemokine gradient and if this gradient is presented by HS on endothelial cells. A gradient was induced by placing a gel containing the chemokine MIP-2 (CXCL2) at a certain distance from the observed blood vessel of wild-type (C57Bl/6, wt) or heparanase transgenic (Hpa-tg) cremaster muscle. Neutrophil-endothelial cell interactions were visualized by real-time and time-lapse intravital microscopy and chemokine gradients were detected by confocal microscopy. Even though similar levels of adherent neutrophils were observed in Hpa-tg and wt mice 60 min after MIP-2 activation, emigration was decreased by 50% in Hpa-tg mice compared to wt. Pretreatment with heparin i.v. competitively bound sequestered MIP-2 in wt mice and thereby completely inhibited leukocyte recruitment. While intraluminal crawling occurred predominantly (76±3%) towards the chemokine gradient in wt mice, in Hpa-tg mice neutrophils showed no preferential crawling direction. In addition, intra- as well as extra-vascular co- localization of MIP-2 and leukocytes was detected in wt mice, while low levels of MIP-2 could be found in the Hpa-tg mice. In conclusion, we found that a chemokine gradient on endothelial cells directed intravascular crawling neutrophils towards the site of infection. Hpa-tg mice revealed a decreased ability to sequester chemokines by the altered endothelial HS, which resulted in random crawling and ultimately decreased neutrophil extravasation.