The metabolic disposition of xenobiotics is of considerable interest to scientists and regulators from pharmaceutical and agrochem. industries. Identification and characterization of metabolites is usually conducted at various stages of compound development using a combination of anal. techniques and synthetic chem. In the early phase of drug development, liquid chromatog. coupled to mass spectrometers is the most frequently used technique to obtain an initial knowledge of the potential in vitro and in vivo metabolites. As the compounds are advanced further into development, other techniques and strategies are used to quantify and better characterize metabolites present in more complex systems such as plant crop extracts and animal-derived biol. matrixes including plasma, urine, bile, and feces. Identification of metabolites in these complex mixtures is often achieved through the use of synthetic reference standards, if available. However, in most cases the metabolite standards are not readily available; hence attempts are made to isolate sufficient amounts of metabolites from these matrixes for addnl. characterization by mass spectrometry and NMR (NMR) spectroscopy. Once the structures are determined, chem. syntheses are conducted to generate sufficient amounts of reference standards The availability of stable- and radio-labeled compounds can also accelerate identification of metabolites, especially in complex biol. matrixes. The overall biotransformation pathways of a compound are proposed after the structures of metabolites are determined This often leads to further investigation into the enzymes responsible for each metabolite formation, species comparison, and potential coverage of human-specific metabolites by toxicol. species. An attempt will be made to demonstrate the utility of the chromatog. procedures as well as the anal. techniques that have been used in the authors' laboratories to isolate and elucidate the structures of metabolites from structurally diverse compounds