A high-performance liquid chromatographic method was developed for the measurement of oxidized and reduced glutathione in biological samples. The method allowed the separation of glutathione (oxidized and reduced) from related thiols (homocysteine, cysteine, cystine, methionine) and other intermediates of glutathione pathways without derivatization or enzymatic reduction. Quantitation was achieved with uv detection. Biological samples were prepared by rapid homogenization in iced KCl followed immediately by deproteinization and acidification with sulfosalicylic acid. Samples were eluted isocratically at 1 ml/min using 0.0025 M sodium phosphate buffer, pH 3.50, containing 0.005 M tetrabutylammonium phosphate (Waters, Milford, MA) and 13% methanol and analyzed on a 30-cm x 3.9-mm C-18 mu Bondapak column and detected with a uv detector at 190 nm. The determination of nanomole levels of glutathione and glutathione disulfide and their separation from other thiols are described.