The unsaturated binding capacities (UBBC) of individual vitamin B12- binding proteins in plasma were measured by a two-step procedure. Transcobalamin II (TC II) was separated by precipitation with ammonium sulfate; the “R”-type binders remaining soluble were then divided into two components by bath separation with anion exchange on DEAE- cellulose. The two R components were designated alpha1-R (TC 1) and alpha2-R (third binder, fetal binder, PV binder, TC III). Ten normal sera were studied by this technique giving a separation into TC III and total plasma R identical to that obtained simultaneously by gel filtration. The mean UBBC of TC II was 969 plus or minus 204 pg of 57 Co B12 per ml of serum. The mean contamination of the precipitated TC III with plasma R was 3%. The UBBCs of alpha 2-R and alpha 1-R were 127 plus or minus 42 and 40 plus or minus 12 pg/ml, respectively. The mean contamination of the R fraction by TC II was 14% as evaluated by gel filtration. By isoelectric focusing it was found that the alpha1-R contained principally those components isoelectric at pH isoelectric at pH of 2.9–3.2, while alpha2-R was made up of those components isoelectric at pH of 3.6 or greater.