Patients on chronic hemodialysis (HD) are at high risk for developing atherosclerosis and cardiovascular complications. Heparanase, a protease that cleaves heparan sulfate (HS) side chains of proteoglycans is involved in extracellular matrix (ECM) degradation, and as such may be involved in the atherosclerotic lesion progression. We hypothesize that heparanase is elevated in HD patients, partly due to its release from primed circulating polymorphonuclear leukocytes (PMNLs), undergoing degranulation. Priming of PMNLs was assessed by levels of CD11b and by the rate of superoxide release. Heparanase mRNA expression in PMNLs was determined by RT-PCR. PMNL and plasma levels of heparanase were determined by immunoblotting, immunofluorescence, and flow cytometry analyses. Levels of soluble HS in plasma were measured by a competition ELISA. This study shows that PMNLs isolated from HD patients have higher mRNA and protein levels of heparanase compared to normal controls (NC), heparanase levels correlate positively with PMNL priming. Plasma levels of heparanase in HD patients were higher than in NC and were further elevated after the dialysis session. In addition, heparanase expression inversely correlates with plasma HS levels. A pronounced expression of heparanase was found in human atherosclerotic lesions. The increased heparanase activity in blood of HD patients results at least in part from degranulation of primed PMNLs and may contribute to acceleration of the atherosclerotic process. Our findings highlight primed PMNLs as a possible source for the increased heparanase in HD patients, posing heparanase as a new risk factor for cardiovascular complications and atherosclerosis.