Prediction of human volume of distribution at steady state (Vss)before first administration of a new drug candidate to humans has become an important part of the drug development process. This study examines the assumptions behind interspecies scaling techniques used to predict human Vss. from preclinical data, namely the equivalency of Vss,u. and/or fut. across species. In addition, several interspecies scaling techniques are evaluated side by side using a set of 67 reference compounds where observed Vss. from rats, dogs, monkeys, and humans were compiled from the literature and where plasma protein binding was determined across species using an ultracentrifugation technique. Species similarity in Vss. or fut. does not appear to be the norm among rats, dogs, monkeys, or humans. Despite this, interspecies scaling from rats, dogs, and monkeys is useful and can provide reasonably accurate predictions of human Vss., although some interspecies scaling approaches were better than others. For example, the performance of the common Vss,u or fut. equivalency approaches using average Vss,u or fut across three preclinical species was superior to allometric scaling techniques. In addition, considering data from several preclinical species, using the equivalency approach, was superior to scaling from any single species. Although the mechanistic tissue composition equations available in the Simcyp® population-based pharmacokinetic simulator did not necessarily provide the most accurate predictions, and the equations used likely need refinement, they still provide the best opportunity for a mechanistic understanding and prediction of human Vss.