Toadfish serum (TFS) offers several advantages over other proteins as the binder in a competitive-binding assay for vitamin B12. It is unaffected by pH changes in the range 5.6-9.4 or by the addition of human serum albumin. Prolonged incubation with charcoal does not disrupt the TFS-cyanocobalamin bond, and the addition of albumin as a protein source in the standard tubes was proven unnecessary. The binding capacity of TFS does not increase significantly with increasing concentrations of cyanocobalamin as does the binding capacity of intrinsic factor, normal serum, or transcobalamin I. A single extract was prepared from each of 44 sera and measured for vitamin B12 content simultaneously by the TFS assay and the conventional microbiologic method using Lactobacillus leichmannii. The values obtained with TFS were in each instance higher than those obtained by the microbiologic assay (p less than 0.001).