We report on the performance of a new test, holotranscobalamin, as compared to well established markers of vitamin B12 deficiency (plasma cobalamins, methylmalonic acid, and homocysteine). Holotranscobalamin was analyzed in 143 samples by a competitive radiobinding assay (Axis-Shield). Employing a cut-off value of 50 pmol/l, holotranscobalamin showed a sensitivity of 1.00 and a specificity of 0.89 as regards discriminating between individuals with test results indicating vitamin B12 deficiency (methylmalonic acid > 0.70 micromol/l and plasma cobalamins < 200 pmol/l, n = 35) and individuals with test results inside the reference intervals (methylmalonic acid < 0.29 micromol/l and plasma cobalamins > or = 200 pmol/l, n = 35). In a group (n = 37) with low plasma cobalamins (< 200 pmol/l) and normal methylmalonic acid (< 0.29 micromol/l), 27 individuals had low holotranscobalamin, and in nine of these individuals plasma homocysteine supported the deficiency state (homocysteine > 15 micromol/l). Holotranscobalamin was low in 12 individuals with increased methylmalonic acid (> 0.40 micromol/l) and normal plasma cobalamins (> or = 200 pmol/l) (n = 36), and plasma homocysteine supported the deficiency state in four of these individuals. We conclude that holotranscobalamin is likely to be a sensitive marker of vitamin B12 deficiency that also has a reasonable specificity. Large-scale clinical studies are warranted in order to clarify the usefulness of holotranscobalamin in the clinical setting.