The study of drug distribution within biological tissue is a key part of the development of new pharmaceuticals. Matrix-assisted laser desorption ionization-mass spectrometric imaging is a powerful new imaging technique that can be used to study the distribution of a diverse range of endogenous and xenobiotic compounds within biological tissue. Here, fundamental aspects of the technique, appropriate instrumentation and applications in the study of xenobiotics and metabolite distribution are described. Sample preparation issues and some of the challenges in data interpretation/handling are also discussed.