Abstract A diminution in the binding of exogenous vitamin B 12 by serum or plasma at pH 1.5 to 2 (acid-resistant binding capacity, ARBC) as compared with the binding capacity at neutral pH (unsaturated vitamin B 12 binding capacity, UBBC) was observed. This phenomenon was found to be attributable to the absence of transcobalamin II (TC II)-associated vitamin B 12 from serum labeled at low pH, as demonstrated by gel chromatography on Sephadex G-200. Further confirmation was obtained by demonstration of a significant correlation between the ARBC and the R binder content, quantitated as resistance to precipitation by ammonium sulfate at a 2M concentration. Serum labeled at acid pH failed to deliver vitamin B 12 to HeLa cells, indicating absence of a “functional” TC II-B 12 complex. The differing vitamin B 12 binding capacities of neutral and acidified material was utilized to fractionate the unsaturated vitamin B 12-binding proteins of serum and plasma. The ARBC was used to measure the R binder content, and TC II was calculated from the difference between UBBC and ARBC. The fractionation procedure was performed on 75 sera and showed increased ARBC in patients with myeloproliferative disorders and decreased ARBC in leukopenia. The content of unsaturated vitamin B 12-binding protein was compared in 75 paired samples of serum and plasma collected from EDTA-anticoagulated blood containing sodium fluoride to inhibit release of granulocyte binders. The ARBC content of fluoridated plasma was significantly lower, due to decreased in vitro R binder release. Plasma also contained less TC II, possibly related to in vitro cellular uptake of this binder in fluoridated plasma.