<jats:title>Abstract</jats:title> <jats:p>Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well-defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem-like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123-low-retention (Rh123low) cells is enriched for stem cell-like activities, including the ability to self-renew and produce nonstem Rh123high progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123low cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123low cells overexpress HIF1α, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin D1 and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123high cells to the Rh123low phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen-activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1α transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123low cells. Moreover, the Rh123low cells became less quiescent and displayed a significant increase in their melanosphere-forming ability. All the above indicates that the Rh123low melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of Rh123low G0 cells promotes the exit of cycling cells from the stem cell compartment.</jats:p>