The competitive inhibition radioisotopic assay of serum vitamin B12 has been adapted for the measurement of cerebrospinal fluid B12. The relatively low CSF B12 levels (1/20 to 1/30 that of serum) necessitated increased sensitivity. This was achieved by using a smaller amount of the labeled B12 for the competitive inhibition, prolonging the incubation time for interaction between B12 and the binding protein, and eliminating the potential for nonspecific binding by maintaining sterility. Proportional dilution studies demonstrated that normal CSF did not contain interfering substances. The range of normal was 8 to 36 pg. per ml. (mean 17.8 pg. per ml.). All of the patients deficient in vitamin B12 studied to date have had reduced CSF B12 values. There was no correlation between CSF B12 and CSF protein or serum B12.