Determination of homocysteine in plasma or serum for evaluation of cobalamin and folate deficiency is becoming an important diagnostic procedure. Accurate, rapid and low cost methods for measuring homocysteine are therefore required. We have improved an HPLC method and made it suitable for clinical application. The more important changes are the addition of an internal standard, mercaptopropionylglycine, and the use of a plasma/serum based calibration material. The method consists of the following steps: reduction of the sample with tri-n-butylphosphine, precipitation of proteins, derivatisation with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate, and HPLC separation followed by fluorescence detection. The linearity of the assays is established and the coefficient of variation is 3.0%. Stability studies show that blood samples must be cooled or centrifuged immediately after venipuncture. The method is useful for evaluation of cobalamin or folate deficiency, especially in patients with normal or moderately depressed cobalamin or folate concentrations in blood.