The difference in ligand localization of manganese in human breast milk, cow's milk and infant formula was investigated. Extrinsic labeling technique was used and the different manganese-binding ligands were separated by gel permeation column chromatography. Manganese was found to bind to different ligands in human milk, cow's milk and infant formula. In human milk, manganese was bound by two high molecular weight proteins, the major one of 407,300 daltons and the minor one of 128,800 daltons. The 407,300-dalton protein was homogeneous with respect to molecular weight and charge and upon saturation with manganese had a metal to protein ratio of 1:1. Cow's milk had three manganese-binding species, with molecular weights of 234,000, 83,200 and less than 1000. The 234,000-dalton manganese-binding fraction was heterogeneous and contained several species with slightly different charge as revealed by DEAE-Sephacel ion-exchange chromatography. Infant formula on the other hand had no high molecular weight manganese-binding species. All the extrinsically added manganese was found in fractions with molecular weight of less than 1000.