To predict volume of distribution at steady-state (Vss), empirical (e.g., allometry) and mechanistic (using physicochemical property data and plasma protein binding) methods have been used. None of these approaches has been able to predict Vss accurately for the total compliment of a wide range of drugs. Therefore, alternative approaches would be of value. This study evaluates the utility of in vitro nonspecific tissue-binding measurements in predicting Vss for a wide range of drugs in rats. Literature as well as proprietary compounds were studied. It was found that in vitro tissue-binding measurements combined with calculated effects of the pH partition hypothesis often predict Vss more accurately than other available mechanistic methods and that this approach can compliment existing methods. The Vss values for some compounds were not accurately predicted using either nonspecific tissue-binding experiments or other available mechanistic methods. The Vss for these drugs may not be describable by nonspecific tissue binding alone; there may be significant specific components to the mechanism of distribution for these drugs, such as pH-dependent uptake into lysosomes (primarily strongly basic drugs), active transport, and/or enterohepatic recirculation. A lack of prediction for certain drugs warrants further investigation into these mechanisms and their application to more accurate prediction of Vss by mechanistic means.