Several human milk oligosaccharides inhibit human pathogens in vitro and in animal models. In an infant, the ability of these oligosaccharides to offer protection from enteric pathogens would require that they withstand structural modification as they pass through the alimentary canal or are absorbed and excreted in urine. We investigated the fate of human milk oligosaccharides during transit through the alimentary canal by determining the degree to which breast-fed infants' urine and fecal oligosaccharides resembled those of their mothers' milk. Oligosaccharide profiles of milk from 16 breast-feeding mothers were compared with profiles of stool and urine from their infants. Results were compared with endogenous oligosaccharide profiles obtained from the urine and feces of age-, parity-, and gender-matched formula-fed infants. In all cases, oligosaccharides were extracted, purified, reduced, and separated into acidic and neutral species; the latter were perbenzoylated and subjected to reversed-phase high-performance liquid chromatography. Structures were determined by mass spectrometry after debenzoylation. Among breast-fed infants, concentrations of oligosaccharides were higher in feces than in mothers' milk, and much higher in feces than in urine. Urinary and fecal oligosaccharides from breast-fed infants resembled those in their mothers' milk. Those from formula-fed infants did not resemble human milk oligosaccharides, were found at much lower concentrations, and probably resulted from remodeling of intestinal glycoconjugates or from intestinal bacteria. Most of the human milk oligosaccharides survived transit through the gut, and some were absorbed and then excreted into the urine intact, implying that inhibition of intestinal and urinary pathogens by human milk oligosaccharides is quite likely in breast-fed infants.