The interactions of reduced glutathione (GSH) with haloacetonitriles (HAN) were carried out under various conditions and the depletion of GSH was determined. Haloacetonitriles reacted directly with glutathione in the absence of tissue extracts. In the presence of cytosolic protein, the depletion of GSH by MCAN was increased, but GSH depletion by DBAN and TCAN was not altered. In the presence of microsomes and a NADPH regenerating system, the consumption of GSH by MCAN and DCAN was increased whereas GSH consumption by DBAN and TCAN was decreased, indicating the biotransformation of HAN to metabolites with different reactivities towards GSH. In the presence of bovine serum albumin, the consumption of GSH by TCAN was decreased while no difference was observed in the depletion of GSH by DBAN. All haloacetonitriles inhibited glutathione-S-transferase activities in vitro. TCAN was the most potent inhibitor, followed by DBAN, BCAN, and MCAN, with DCAN was the least reactive.