An additional biliary glutathione (GSH) conjugate of the anilinoacridine anti-tumour agent amsacrine (4'-(9-acridinylamino)methanesulphon-m-anisidide, NSC 249992) has been identified in bile collected from male Wistar rats by cannulation of the common bile duct and from male BDF1 mice by removal of the gall bladder after treatment with amsacrine. The presence of this conjugate, at the 6'-position of the anilino ring, has been confirmed by liquid secondary ion (LSI) mass spectrometric analysis of selected biliary metabolites separated by high-performance liquid chromatography. The two major metabolites each gave a daughter ion spectrum which was diagnostic for either 5'- or 6'-GSH conjugation. This pattern was confirmed by comparison with LSI mass spectral data obtained from authentic chemical standards formed on reaction of the quinone diimine derivative of amsacrine with methanethiol or mercaptoethanol.