The clinical importance of a reliable human serum vitamin B 12 assay to aid the diagnosis of pernicious anemia (PA) cannot be overemphasized. Our review of the literature indicates that a reference method for the quantitation of serum vitamin B 12 (serum B 12) with the required accuracy, precision and rapidity has not been reported to-date. Controversies, debates and criticisms over human serum B 12 assays (especially commercial kits) have been commonplace. Various methods of quantitation of B 12 reviewed in this communication include: microbiological, radioisotopic, high-performance liquid chromatography (HPLC) and the most recent radioimmunoassay (RIA) techniques. This review attempts to provide awareness of the limitations of these methods and establishes the base for eventual development of a B 12 reference method in our laboratory.