Human milk oligosaccharides (HMO) have been paid much attention due to their beneficial effects observed in vitro, e.g. prebiotic, anti-infective, and anti-inflammatory properties. However, in vivo investigations with regard to HMO metabolism and functions are rare. The few data available indicate that HMO are absorbed to a low extent and excreted via urine without noteworthy modifications, whereas the major proportion reaches the infant's colon undigested. Via intrinsic (13)C-labeling of HMO during their biosynthesis in the mammary gland of ten lactating women, we were able to follow the fate of (13)C-labeled oligosaccharides (OS) from their secretion in milk to the excretion in the urine of their breastfed infants. To a certain extent, we could therefore discriminate between original HMO and non-labeled OS derived from degradation of HMO or endogenous glycoconjugates. By means of our novel, rapid matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based approach, we found a homogeneous time pattern of isotopomer enrichment in milk among all subjects and between single OS species. In contrast, the time curves from infants' urine varied strongly between individuals and OS species, though the overall MALDI-TOF MS profile resembled those of the mothers' milk. Our data suggests that neutral HMO might be processed and/or utilized differentially after or upon absorption from the gut, as deduced from their structure-dependent variation in the extent of tracer enrichment and in the retention times in the infant's organism. This sheds new light on the role of HMO within the infant's body, beyond the intestine and its microbiota alone.