Melanomas contain subsets of cancer stem-like cells with tumor-initiating capacity. The frequency of these cells in the tumor is still a topic of debate. We investigated the phenotypic plasticity of cancer cells grown as melanospheres to elucidate the influence of the microenvironment on some features of melanoma stem-like cells. Cells from surgical specimens of nodular melanoma were grown as anchorage-independent melanospheres in a stem cell medium and as adherent monolayer cultures in the presence of serum. Proliferation and viability were measured by cell counting and an acid phosphatase assay; surface marker expression was evaluated by flow cytometry, and the clonogenic potential of single cells was assessed by growth in soft agar. Patient-derived melanoma cells could be maintained in cell culture for more than 16 months when grown as melanospheres. In the presence of serum, melanospheres completely changed their growth characteristics and formed adherent monolayers. The transition from melanospheres to monolayers was accompanied by an apparent loss of clonogenic potential, an increased proliferation rate, and altered expressions of cell surface markers ABCB5, CD133, and CD49f. These changes, however, were reversible. Compared with adherent monolayer cultures, melanospheres are enriched in cells with clonogenic potential, reflecting the self-renewing capacity of cancer stem-like cells. This clonogenic potential can be lost and regained depending on the growth conditions. Our results demonstrate how easily melanoma cells change their function upon exposure to external stimuli and suggest that the frequency of melanoma stem-like cells strongly depends on the microenvironment.