The use of animals in science is a widespread practice, despite growing concern about its moral justification and scientific relevance. In this scenario, the Three Rs concept might be considered to be a motivation for the establishment of a new scientific approach to the use of experimental animals and to research itself. The main objective of this survey-based study was to identify the level of knowledge about this concept among lecturers (i.e. tenure-track professors) and postgraduate students in the physiological and pharmaceutical sciences in Brazilian universities. A questionnaire was completed by 185 lecturers from 16 universities, and 140 postgraduate students from five of these universities. The results indicate that the concept of the Three Rs is widespread among lecturers and students in the areas of physiology and pharmacology, throughout Brazilian universities, but that its interpretation generally attributes more importance to refinement than to reduction and replacement.