Receiver operating characteristics (ROC) analysis has been used in many medical imaging applications during the past decade. With the recent expansion of the ROC methodology to multi-disease studies, several reports have begun to address both the theoretical and experimental design issues associated with such studies. While the appropriate selection, classification, and verification of actually positive cases is carefully addressed in the literature, similar considerations are rarely given to the selection of actually negative controls for these studies. In this paper, theoretical considerations and experimental data are provided to demonstrate the significance of this very issue.