Gelatinase B is a matrix metalloproteinase (MMP-9) expressed under strict control by many cell types including neutrophils, monocytes, macrophages, and tumor cells. MMP-9 is a key mediator in the physiological maintenance of the extracellular matrix both in tissue remodeling and development, while uncontrolled enzyme activity contributes to pathologies such as cancer and inflammation. Neutrophils release MMP-9 from granules in response to IL-8 stimulation. Human MMP-9 has three potential N-linked glycosylation sites and contains a Ser/Pro/Thr rich domain, known as the type V collagen-like domain, which is expected to be heavily O-glycosylated. Indeed, approximately 85% of the total sugars on human neutrophil MMP-9 are O-linked. This paper presents the detailed analysis of picomole amounts of these O-glycans using a novel HPLC-based strategy for O-glycan analysis that provides linkage and arm specific information in addition to monosaccharide sequence. The initial structural assignments were confirmed using HPLC with online MS/MS fragmentation analysis. Twelve sugars were identified that contained from two to nine monosaccharide residues. Most of these contained type 2 core structures with Galbeta1-4GlcNAc (N-acetyl lactosamine) extensions, with or without sialic acid or fucose. The O-glycans were modeled using the oligosaccharide structural database. On the basis of the structure of gelatinase A (MMP-2), a model of MMP-9 suggests that the type V collagen-like domain in gelatinase B is located on a loop remote from the active site. Fourteen potential O-glycosylation sites are multiply presented on this loop of 52 amino acids. Many of the O-glycans identified contain terminal galactose residues that may provide recognition epitopes. Importantly, heavy glycosylation of this loop region, absent in gelatinase A, has considerable implications for the domain organization of MMP-9.