In radioassays for serum vitamin B-12, the separation of free and bound vitamin is usually made with charcoal absorption. The specificity of this separation depends on the amount of charcoal and the protein content and constitution of the medium. The large difference in protein concentration between the samples for the dilution curve and the serum samples introduces an uncontrolled variable in the test. In order to equalize the experimental circumstances, the standard dilutions were made in serum freed from vitamin B-12 after boiling the serum for 20 min in a 4-fold dilution with glutamic acid buffer at pH 3.3 and subsequent passage over a CH-Sepharose 4-B column complexed with hog intrinsic factor (IF). The vitamin B-12-binding capacity of such an affinity column prepared from 1 g CH-Sepharose and 20 mg IF, suffices for the absorption of vitamin B-12 in 3000 ml serum from which 300 series of vitamin B-12 standard solutions can be made. Our first results with this method confirm that the charcoal absorption radioassay has become more accurate by the use of vitamin B-12-free serum in the standard dilutions.