The radioisotopic saturation analysis assay has demonstrated its applicability to the determination of serum vitamin B12 levels; however, several potential problems have existed in the assay. These are the basis of this report. It has been demonstrated that modification of the conditions of the assay will improve the separation of endogenous B12 from its binding proteins and reduce potential nonspecific binding of the liberated B12. Pooled, Millipore-filtered, normal serum serves as an excellent binder for the assay. The problems of alternate binders are reviewed. Since occasional recovery values varied, an extraction control has been included. Finally, the problems of identification of radiochemical purity of the 57CoB12 are reviewed and a method of detection of impurities is described.