1. After fractionation of the vitamin B12-binding proteins of ten normal sera the components containing transcobalamin II and R-type binders of vitamin B12 respectively were studied for endogenous vitamin B12 content by two distinct systems of vitamin B12 assay.
2. The measurements of total serum vitamin B12 by either bioassay with Euglena gracilis or a radioisotope dilution assay agreed closely.
3. The native vitamin B12 carried by transcobalamin II was higher as measured by bioassay than by isotope dilution assay.
4. The presence of the transcobalamin II fraction of human serum altered the key reaction between the binding reagent of the isotope dilution assay, so that this assay failed to measure vitamin B12 quantitatively.
5. Probably, the mean fraction of plasma vitamin B12 carried by transcobalamin II is in the range 20–30%.