The structure of two biliary metabolites of 4-cyano-N,N-dimethyl aniline (CDA) contained in whole rat bile have been studied in detail by NMR at 400 MHz. A 4-cyano-N-methyl glutathione-N-aniline conjugate was identified as a biliary metabolite of CDA using relatively simple 1H NMR techniques. Isotopically 13C labelled CDA was used to generate 13C labelled xenobiotic conjugates. Our use of 1H/13C heteronuclear NMR techniques, in particular a 13C-selective HMQC-TOCSY experiment, allowed a N-beta-glucuronide conjugate, a previously unknown biliary metabolite of CDA, to be identified. Bile samples obtained from both the isolated perfused rat liver and the rat liver in situ were analysed.