Heparanase (HPSE1) is known to be involved in mechanisms of metastatic tumour cell migration. This enzyme selectively cleaves heparan sulfate proteoglycans (HSPG), which are ubiquitously expressed in mammals and are known to be involved in regulating the activity of an array of inflammatory mediators. In the present study, we have investigated the effects of human recombinant heparanase, the inactive precursor of this enzyme (pro-heparanase) and enzymatically inactivated heparanase, on inflammatory cell recruitment in the rat and on human leukocyte-endothelial adhesion in vitro. Intraperitoneal injection of heparanase (500 μg) induced a significant inflammatory cell infiltrate in the rat, assessed by peritoneal lavage four hours later. Intravital microscopy of the mesenteric microcirculation of anaesthetized rats showed an increase in rolling and adherent cells in post-capillary venules that was sensitive to heparin, a non-selective inhibitor of heparanase activity. In vitro, heparanase augmented the adhesion of human neutrophils and mononuclear cells to human umbilical vein endothelial cells in a concentration-dependent manner. Pro-heparanase had similar effects to the active enzyme with respect to leukocyte accumulation in the peritoneal cavity and adhesion in vitro. However, heat-inactivated heparanase induced cell adhesion in vitro but was without effect in vivo. Together, these data indicate a role for heparanase in inflammatory cell trafficking in vivo that appears to require enzymatic activity.