Conventional cytotoxic anticancer drugs that target all rapidly dividing cells are nonselective in their mechanism of action, because they disrupt essential components that are crucial to both malignant and proliferating normal cells. Instead, targeting cellular functions that are distinctly different between normal and cancer cells may provide a basis for selective killing of tumor cells. One such strategy that is still largely unexplored is to utilize the relatively higher negative mitochondrial membrane potential in carcinoma cells compared with adjacent normal epithelial cells to enhance accumulation and retention of cytotoxic lipophilic cations in the former. In this study, the anticancer activities of a new class of rosamines with cyclic amine substituents and their structure-activity relationships were investigated. From an in-vitro cell growth inhibition assay, 14 of the rosamines inhibited the growth of human leukemia HL-60 cells by 50% at micromolar or lower concentrations. Derivatives containing hydrophilic substituents had less potent activity, whereas aryl substitution at the meso position conferred extra activity with thiofuran and para-iodo aryl substitutions being the most potent. In addition, both compounds were at least 10-fold more cytotoxic than rhodamine 123 against a panel of cell lines of different tissue origin and similar to rhodamine 123, exhibited more cytotoxicity against cancer cells compared with immortalized normal epithelial cells of the same organ type. In subsequent experiments, the para-iodo aryl substituted rosamine was found to localize exclusively within the mitochondria and induced apoptosis as the major mode of cell death. Our results suggest that these compounds offer potential for the design of mitochondria-targeting agents that either directly kill or deliver cytotoxic drugs to selectively kill cancer cells.