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This chapter presents an overview of the ethical, legal, and scientific reasons that mandate the alleviation of animal pain, drawing attention to the principles of the Three Rs (3Rs; replacement, refinement, and reduction) and the central role of refinement in the humane care and use of laboratory animals. It includes discussion of the fundamental concepts of the experience of pain and factors that affect pain aversiveness. It focuses on the potential causes of pain in research animals while broadly considering evidence of pain in vertebrates. It concludes with a discussion of the particular circumstances that may justify pain in laboratory animals.