Disialylgalactosylgloboside (DSGG), defined by monoclonal antibody RM2, is a renal cell carcinoma (RCC)-associated antigen which mediates adhesion of RCC TOS-1 cells to certain lung tissue target cells. This adhesion process may initiate preferential lung metastasis of RCC. Ganglioside GM3 is a B16 melanoma-associated antigen which similarly adheres to target cells and promotes consequent metastasis. In view of the close association of GM3-enriched microdomain with transducer molecules c-Src, Rho A, and FAK in B16 cells, we investigated the organizational status of DSGG in RCC cell line TOS-1, with the following results: i) DSGG, but not monosialylgalactosylgloboside, showed extensive clustering at the TOS-1 cell surface; ii) a low-density membrane fraction isolated from TOS-1 cells contained >95% of cellular DSGG, although protein content in this fraction was <1% of total cellular protein; iii) this fraction contained c-Src, Rho A, and FAK, but not H-Ras; iv) c-Src and Rho A were co-immunoprecipitated with DSGG through anti-DSGG mAb RM2 (IgM) affixed to a column. These observations indicate that DSGG is clustered in RCC, as typified by TOS-1 cells, to form a microdomain in which it is closely associated with c-Src, Rho A, and FAK, and may constitute a functional unit as has been observed for GM3 with transducer molecules in B16 cells. The functional organization of such units may be essential in determining malignant properties of RCC cells.