Questions persist about the nature and number of cells with tumor-propagating capability in different types of cancer, including melanoma. In part, this is because identification and characterization of purified tumorigenic subsets of cancer cells has not been achieved to date. Here, we report tumor formation after injection of single purified melanoma cells derived from three novel mouse models. Tumor formation occurred after every injection of individual CD34⁺p75⁻ melanoma cells, with intermediate rates using CD34⁻p75⁻ cells, and rarely with CD34⁻p75⁺ cells. These findings suggest that tumorigenic melanoma cells may be more common than previously thought and establish that multiple distinct populations of melanoma-propagating cells (MPC) can exist within a single tumor. Interestingly, individual CD34⁻p75⁻ MPCs could regenerate cellular heterogeneity after tumor formation in mice or multiple passages in vitro, whereas CD34⁺p75⁻ MPCs underwent self-renewal only, showing that reestablishment of tumor heterogeneity is not always a characteristic of individual cells capable of forming tumors. Functionally, single purified MPCs were more resistant to chemotherapy than non-MPCs. We anticipate that purification of these MPCs may allow a more comprehensive evaluation of the molecular features that define tumor-forming capability and chemotherapeutic resistance in melanoma. Cancer Res; 70(1); 388–97