c-Ski has been known to be phosphorylated at serine residue(s), which results in slower migration of c-Ski in SDS-polyacrylamide gel electrophoresis. The position(s) of phosphorylation, however, has not been determined. In the present study, we identified a phosphorylation site of c-Ski which affects its electrophoretic motility as serine 515 using MALDI-TOF mass spectrometry. A phosphorylation-resistant mutant, c-Ski S515A, did not exhibit a phosphatase-sensitive band shift. In addition, we confirmed that endogenous c-Ski is phosphorylated at serine 515, using a specific antibody. The phosphorylation status of c-Ski, however, does not appear to affect its stability or effects on TGF-β signalling. Identification of the phosphorylation site of c-Ski would allow us further examination of physiological significance of c-Ski phosphorylation.