Cripto-1 (CR-1) protein function differs according to cellular or extracellular expression. In this study, we explore the significance of cell surface CR-1 expression in human melanoma cells. Cell surface CR-1-expressing human melanoma cells (CR1-CS+) were selected by fluorescence-activated cell sorting (FACS) cell sorting and grown in vitro and in vivo in nude mice to study their growth characteristics. The CR1-CS+ melanoma cells were found to express increased levels of Oct4, MDR-1 and activated c-Src compared with cells lacking this subpopulation (CR1-CS-) or unsorted cells, used as control. CR1-CS+ show reduced proliferation rates and diminished spherical colony formation compared with control cells when cultured in vitro. Orthotopic injections of CR1-CS+ in nude mice formed slow growing tumors with histologic variability across different areas of the CR1-CS+ xenografts. CR-1-expressing cells from first generation CR1-CS+ tumors showed significantly increased tumor-forming rate and aggressiveness following subsequent transplants in nude mice. These data demonstrate that within a heterogeneous melanoma cell population there resides a slow proliferating, cell surface CR-1-expressing subpopulation capable of giving rise to a fast growing, aggressive progeny that may contribute to disease recurrence and progression.