In previous papers, we have offered a strategic framework regarding metabolites of drugs in humans and the need to assess these in laboratory animal species (also termed Metabolites in Safety Testing or MIST; Smith and Obach, Chem. Res. Toxicol. (2006) 19, 1570-1579). Three main tenets of this framework were founded in (i) comparisons of absolute exposures (as circulating concentrations or total body burden), (ii) the nature of the toxicity mechanism (i.e., reversible interaction at specific targets versus covalent binding to multiple macromolecules), and (iii) the biological matrix in which the metabolite was observed (circulatory vs excretory). In the present review, this framework is expanded to include a fourth tenet: considerations for the duration of exposure. Basic concepts of pharmacology are utilized to rationalize the relationship between exposure (to parent drug or metabolite) and various effects ranging from desired therapeutic effects through to severe toxicities. Practical considerations of human ADME (absorption-distribution-metabolism-excretion) data, to determine which metabolites should be further evaluated for safety, are discussed. An analysis of recently published human ADME studies shows that the number of drug metabolites considered to be important for MIST can be excessively high if a simple percentage-of-parent-drug criterion is used without consideration of the aforementioned four tenets. Concern over unique human metabolites has diminished over the years as experience has shown that metabolites of drugs in humans will almost always be observed in laboratory animals, although the proportions may vary. Even if a metabolite represents a high proportion of the dose in humans and a low proportion in animals, absolute abundances in animals frequently exceed that in humans because the doses used in animal toxicology studies are much greater than therapeutic doses in humans. The review also updates the enzymatic basis for the differences between species and how these relate to MIST considerations.