Human milk (HM) is unique compared with the milk of other species in that nonprotein nitrogen (NPN) constitutes 20-25% of the total N. The NPN fraction consists of a diverse group of compounds with molecular masses less than 10,000 Da (in the picogram to microgram per milliliter range), which have only been partially characterized. We developed a methodology to separate and concentrate the NPN fraction for further analysis. NPN was initially separated from other milk components by Sephadex G-25 gel filtration. Further isolation and separation was carried out by fast protein liquid chromatography gel filtration and ion-exchange chromatography. Molecular masses of unknown peaks were determined by using known molecular mass markers and standards. The methodologies developed lead to the discrete separation of NPN from other milk compounds and can be particularly valuable for isolating peptides in HM.