The heat-stable antigen (HSA) is a marker of hematopoietic differentiation in both the B and T cell lineages. The antigen is recognized by a series of monoclonal antibodies which includes J11d, M1/69 and B2A2, and in addition YBM5.10.4. We show here that all these antibodies recognize the same antigenic determinant which is expressed on a variably glycosylated membrane protein. Tunicamycin experiments show that the antigen is not carbohydrate in nature as it is expressed on two unglycosylated protein core molecules of molecular mass ca. 20 kDa and 17 kDa. Furthermore, the antigenic determinant appears to be lost following phosphatidylinositol-specific phospholipase C cleavage. Although the molecular mass of HSA appears to be heterogenous on cells of different lineages, these variations in size appear to be due primarily to differences in the extent of N-linked glycosylation, since both protein core molecules were found in all cell types investigated which express the antigen. These findings have important implications for the structure and function of this antigen and its role in hematopoietic development.