Four different methods for determination of “free” vit B12 and the fraction “bound” by the serum proteins have been compared. The serum binding capacity for vit B12. at varying concentrations of total vit B12 has been determined with the same methods. None of the methods showed correlation between original serum vit B12 concentrations and binding of added vit B12. When binding capacity was determined by dialysis and by using uptake of vit B12 by wild type E. coli, there was good agreement. This showed a mean binding capacity of 1350 pg vit B12/ml serum when total vit B12 concentration was below 10,000 pg/ml. Amount of vit B12 unavailable for L. leichmannii increased almost proportionally with total concentration of vit B12. Amount of vit B12 unavailable for E. gracilis was almost constant, irrespective of total concentration of vit B12.