Abstract: For determination of total sulfhydryls, plasma or urine was incubated with dithiothreitol (I) for 15 min at 37°C, mixed with sulfosalicylic acid and centrifuged. For determination of free sulfhydryls, acid-pptd. plasma supernatant solution was mixed with boric acid, NaOH solution and I and incubated at 37°C for 15 min. Analysis was by HPLC on a column (10 cm x 3.2 mm) of Brownlee Velosep RP-18 (3 µm) with a mobile phase (0.8 ml/min) of 16 mM NaH2PO4/19 mM H3PO4/8 mM octyl sulfate/4.3% methanol (pH 2.4). Post-column derivatization was with 10 mM 4,4'-dithiodipyridine in 0.01 M HCl/methanol (97:3) and 300 mL of 0.3 M Tris/1 mM EDTA (pH 8.5) followed by detection at 324 nm. The limit of detection was 50 nM-homocysteine and the calibration graph was linear for 0.2-100 µm-homocysteine. The intra- and inter-assay RSD for plasma were 1.1-5.5% (n = 7) and 2.5% (n = 22), respectively. Recoveries were 94.0, 97.1, 86.5 and 112.3% for cysteine, homocysteine, glutathione and cysteinylglycine, respectively.