Human milk glycosidic enzymes and biologically active glycoconjugates incubate together in the breast between the time of synthesis of milk and the next feed, and during storage of expressed milk. The degree to which the glycoconjugates of human milk are modified by glycosidases was investigated. Human milk was freshly obtained in the laboratory from four women. The activities of alpha-L-fucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-glucosidase, N-acetyl-beta-hexosaminidase, beta-D-glucuronidase, and neuraminidase in were determined; fucosidase and hexosaminidase displayed the highest activity. Free fucose, N-acetylneuraminic acid (NANA) and N-acetylhexosamines were also measured by gas chromatography and gas chromatography/mass spectroscopy. Incubation of milk samples for 2 to 16 hours at 37 or 20 degrees C, but not at 4 degrees C, increased the amounts of fucose, NANA, and N-acetylhexosamines, consistent with enzymatic release by the endogenous glycosidases. The milk contained small amounts of free sugars, whose concentrations were used to determine the upper limits of postsynthetic modification of glycoconjugates during the residence time of milk in the breast of these four donors. These data indicate that under typical conditions glycoconjugate degradation in milk is modest.