Derivatives of vitamin B(12) (cobalamin, Cbl) are required for activity of the mitochondrial enzyme L-methylmalonyl-CoA mutase and the cytoplasmic enzyme methionine synthase in human cells. We recently described a putative novel Cbl-binding protein in crude mitochondrial fractions isolated from cultured fibroblasts. The amount of Cbl bound to this protein varied in fibroblasts from patients with different genetic defects affecting cobalamin metabolism. We have now identified this protein as the cobalamin transport protein transcobalamin (TC) by its binding to anti-TC antibodies and mass spectrometry, and suggest that its presence in crude mitochondrial fractions was the result of lysosomal contamination. Increased Cbl bound TC levels were confirmed in whole cell extracts in at least one cell line from both the cblB and mut classes of inborn errors of cobalamin metabolism.