Hyaluronan is a ubiquitous glycosaminoglycan of high molecular weight that acts as a structural component of extracellular matrices and mediates cell adhesion. There have been numerous recent reports that fragments of hyaluronan have different properties compared to the intact molecule. Though many of these results may be genuine, it is possible that some activities are due to minor components in the preparations used. Therefore, it is important that well-characterized and highly purified oligosaccharides are used in cell biological and structural studies so that erroneous results are avoided. We present methods for the purification of hyaluronan oligomers of defined size using size exclusion and anion-exchange chromatography following digestion of hyaluronan with testicular hyaluronidase. These preparations were characterized by a combination of electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight analysis, and fluorophore-assisted carbohydrate electrophoresis. Hyaluronan oligomers ranging from tetrasaccharides to 34-mers were separated. The 4- to 16-mers were shown to be homogeneous with regard to length but did contain varying amounts of chondroitin sulfate. This contaminant could have been minimized if digestion had been performed with medical-grade hyaluronan rather than the relatively impure starting material used here. The 18- to 34-mer preparations were mixtures of oligosaccharides of different lengths (e.g., the latter contained 87% 34-mer, 10% 32-mer, and 3% 30-mer) but were free of detectable chondroitin sulfate. In addition to oligomers with even numbers of sugar rings, novel 5- and 7-mers with terminal glucuronic acid residues were identified.