Evaluation of experimental drugs in animals for effects on critical organ systems allows identification of functional signals of efficacy and safety that can be subsequently monitored in human clinical trials. The International Conference on Harmonization (ICH), 'Guidelines on Safety Pharmacology', finalized in 2000, defined critical organ systems (cardiovascular, respiratory and central nervous system) and functions to be evaluated, and points to consider for study design and conduct. The new Safety Pharmacology guidelines recognise that while in vitro studies of molecular targets (enzymes, receptors, ion channels, etc.) suggest mechanisms by which chemicals might impact organisms, organ functions are complex, integrative, and most usefully evaluated in their totality, in intact and ideally unanesthetized animal models. Signals identified from safety pharmacology studies are candidates for biomarkers, which when established/validated, can enhance cross-species-based risk assessments and risk management in clinical trials. This review will briefly trace the origins of modern safety pharmacology and discuss practical issues related to the identification and application of signals generated from safety pharmacology studies. The QT interval from the electrocardiogram is currently the 'most validated' of those signals ('biomarkers') generated from safety pharmacology studies and is presented as an example of the utility and the difficulties faced by safety pharmacologists attempting to predict risk in humans based upon physiological measurements conducted in animal studies.