Polymorphonuclear leucocytes (PMNs) accumulate at inflammatory sites and contribute to host defence, regulation of the inflammatory process, and also to tissue injury. Upon activation, these cells release the serine proteases elastase, cathepsin G, and proteinase 3 that are involved in multiple processes such as microbicidal activity, penetration of PMNs through endothelium and adjacent connective tissue to inflammatory sites, and processing of various cytokines. Here, we compared the three serine proteases for their release from PMNs and their ability to interact with resting PMNs and the highly sulphated glycosaminoglycan heparin. Unlike elastase, proteinase 3 and cathepsin G were released from resting PMNs as evidenced by flow cytometry, confocal fluorescence microscopy, and activity measurements. While proteinase 3 binds heavily to surface targets on vital PMNs, cathepsin G and elastase interact preferentially with sulphated glycosaminoglycans. These data revealed a differentiated picture about the individual functions of the PMN serine proteases during inflammatory response.