A 36-kDa protein was isolated by affinity chromatography using Cymelarsan, an arsenical drug currently used in African trypanosomiasis treatment, as ligand. This protein was identified as glycerol-3-phosphate dehydrogenase. Trypanosomal glycerol-3-phosphate was bound covalently, whereas its counterpart from rabbit muscle bound by ionic interaction. Arsenical drugs inhibit the enzyme in a dose-dependent manner. Oxidation of cysteine residues protects against inactivation without significantly diminishing enzymic activity. Drug concentrations giving 50% inhibition of the dehydrogenase activity were determined for the enzyme from both Trypanosoma brucei and rabbit and indicate a higher sensitivity of the trypanosomal enzyme to arsenical drugs and thiol reagents. MS was used to identify residues of glycerol-3-phosphate dehydrogenase bound by Cymelarsan; they are not conserved in the mammalian enzyme.