The most convenient route of drug administration is peroral. To reach their target, drug molecules must be absorbed from the gastrointestinal tract and enter the systemic circulation in sufficient quantities. For this reason, understanding and anticipating the mechanisms and factors affecting gastrointestinal absorption and metabolism are of the utmost importance in developing new drugs. In contrast to drugs, which are administered intentionally for therapeutic reasons, chemical residues in food and other matrices enter the body unintentionally. Hence, in this case, a low systemic availability would be advantageous. For many reasons, but particularly because of financial and ethical (reduced used of animals) considerations, in vitro and ex vivo approaches to this problem have been pursued over the last few years. The use of in vitro methods, however, inherently creates questions about the validity of extrapolation to the in vivo situation. The purpose of this report is to review the current status of the field and to identify major gaps in our knowledge. Currently, there are a number of in silico, in vitro, cultured cell-based and ex vivo approaches available to predict the cell permeation, absorption and gastrointestinal metabolism of molecules. Some strengths and weaknesses of these approaches are presented, together with a discussion of genetic, environmental, physiological and pathological factors responsible for interspecies and inter-individual variability in these processes. Recent advances in our understanding of active processes such as gut epithelial transporters, involved in absorption, and drug-metabolising enzymes, responsible for intestinal presystemic metabolism, are highlighted. Some major research priorities are identified, including the need for high-quality, information-rich databases against which testing methods being developed can be prevalidated and validated. Preclinical drug development is changing rapidly, and the role of in vitro and ex vivo approaches in this process is becoming increasingly more important. Methods available now are very useful in the drug discovery and development process, including lead compound selection and optimisation and in the design of very early clinical studies, but whether any of them will eventually obviate the need for clinical trials of bioavailability is still very debatable and will require their full validation. It is clear, however, that the results from such in vitro tests are important in shaping drug discovery and the early preclinical drug development process. For other environmental, industrial and household chemicals to which humans are exposed, in particular new chemicals, results from in vitro studies might be the only source of information concerning systemic availability.