The vitamin B12 content and binding capacity of cow's milk protein fractions after equilibration with excess 60Co-labeled vitamin B12 in borate buffer, pH 9.0, were determined by microbiological assay using Lactobacillus leichmannii and by assay for radioactivity. The distribution of vitamin B12 between the various proteins of milk was ubiquitous but a higher concentration was demonstrated in the whey proteins. The isoelectrically prepared casein fraction contained 98 µµg of vitamin B12/mg of protein; β-lactoglobulin, 870; α-lactalbumin, 90; blood serum albumin, 80; proteose-peptone, 370; fat-globule membrane proteins, 82; and non-protein nitrogen — converted to an equivalent amount of protein, 2,200. The vitamin B12 content of the casein complex was distributed between α- and β-caseins: 41 and 20 µµg/mg of protein, respectively. Further fractionation of α-casein into αs- and k-caseins yielded values for vitamin B12 of 5 and 10 µµg/mg of protein, respectively. Apparently, the vitamin was released during the subfractionation of the casein complex. The amount of 60Co-labeled vitamin B12 adsorbed by various milk proteins upon equilibration with excess 60Co-labeled vitamin B12 was 850 µµg/mg for β-lactoglobulin, 690 for α-lactalbumin, 773 for casein, 1580 for α-casein, 915 for αs-casein and 2490 for the proteosepeptone fraction. In 8 M urea, β-lactoglobulin bound vitamin B12 at a higher level (e.g., > 20%).