Three different monoclonal anti-H-2Kk antibodies, 27R9, 30R3 and 11-4 were examined for the biochemical nature of the antigenic determinants they recognize. When these were compared on the basis of their sensitivity to pronase and various glycosidases, 27R9 was shown to bind to protein-defined H-2Kk antigens, while 30R3 and 11-4 bound to H-2 antigens defined by carbohydrate. From sugar inhibition studies, and treatments with specific glycosidases, d-mannose appears to be the immunodominant sugar involved in the antigenic site recognized by 30R3, while several sugars, namely sialic acid, d-mannose and α-and β-linked d-galactose would appear to be components of the antigenic site bound by 11-4. The carbohydrate determinants appear to be present on glycolipid molecules, since both the 30R3 and 11-4 antibodies could be inhibited by glycolipid extracts from spleen cells of the appropriate H-2 haplotype, as well as from several other strains of mice previously shown to be cross-reactive targets for these antibodies. This finding is supported by evidence that the molecule carrying the protein-defined antigen is distinct from that carrying the carbohydrate-defined antigens. The results are discussed in the light of current information on the nature of glycolipid Ia antigens, as well as the role of H-2 antigens in T-cell interactions.