Many human milk glycoconjugates (glycolipids, glycoproteins, mucins, glycosaminoglycans) and oligosaccharides are biologically active, and their activity depends on the precise structure of the glycan. The sugars on the terminus of the glycan are vulnerable to cleavage by glycosidases. Because glycoconjugates incubate together with endogenous glycosidases in the breast between feedings, and in expressed milk during storage, the presence and activity of glycosidases in human milk was investigated. alpha-L-Fucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucosidase, N-acetyl-beta-hexosaminidase, beta-D-glucuronidase, and neuraminidase were measured in milk samples from 4 donors by use of synthetic fluorogenic glycosides; fucosidase and hexosaminidase displayed the highest activity. The catabolic activity of the major glycosidases was confirmed by measuring the corresponding free sugars in milk. Free fucose, N-acetylneuraminic acid, and N-acetylhexosamines were measured and their identities were confirmed by high-performance liquid chromatography, gas chromatography, and gas chromatography-mass spectrometry. Incubation of samples for 16 hr at 37 degrees C and 20 degrees C, but not at 4 degrees C, resulted in time-dependent increases in the amount of free fucose, N-acetylneuraminic acid, and N-acetylhexosamines, consistent with their enzymatic release by the endogenous glycosidases. Stored frozen milk had the same levels of these sugars as did samples analyzed immediately after collection, indicating that glycosidases are inactive in the frozen milk. Samples analyzed immediately after collection contained only small amounts of free sugars, suggesting that glycoconjugate degradation during the typical residence time of milk in the breast is modest.