Mannosylerythritol lipid (MEL), a novel extracellular glycolipid from yeast, was found to inhibit the proliferation of mouse melanoma B16 cells in a dose-dependent manner and to induce the apoptosis of B16 cells at concentrations higher than 10 microm (Zhao, X., Wakamatsu, Y., Shibahara, M., Nomura, N., Geltinger, C., Nakahara, T., Murata, T., and Yokoyama, K. K. (1999) Cancer Res. 59, 482-486). We show here that exposure of B16 cells to MEL (5 microm) for 2 days resulted in an increase of the levels of differentiation-associated markers of melanoma cells such as melanogenesis and tyrosinase activity, which were accompanied by morphological changes. The MEL-induced differentiation of B16 cells at this concentration was closely associated with arrest of the cell cycle at G(1) phase, but no significant population of apoptotic cells was identified. Expression of protein kinase Calpha (PKCalpha) was enhanced after exposure of B16 cells to MEL for 48 h. Antisense oligodeoxynucleotides against the mouse gene for PKCalpha prevented MEL-induced melanogenesis in B16 cells. Conversely, the effects of the expression of a constitutively active form of PKCalpha mimicked the effects of MEL on B16 cells. These data suggest that MEL, a yeast-derived glycolipid, triggers the differentiation of B16 melanoma cells through a signaling pathway that involves PKCalpha.