Carbohydrate-carbohydrate interactions between GM3 and Gg3 were investigated using surface pressure-area (pi-A) isotherms of glycolipid monolayers in contact with glycoconjugate polystyrenes at the air-water interface. The GM3 monolayer was greatly expanded by the Gg3-trisaccharide (GalNAcbeta1-4Galbeta1-4GIc)-bearing glycoconjugate polystyrene [PN(Gg3)] and moderately expanded by lactose-bearing polystyrene, while little or no expansion was observed with GalLac (Galbeta1-4Galbeta1-4GIc)- or cellobiose-bearing polystyrenes or Gg3-trisaccharide-bearing styrene monomer. The PN(Gg3)-induced expansion of,pi-A isotherms of sphingolipid monolayers was maximal with GM3 and decreased in the following order: (KDN)GM3 > GM2 approximate to LacCer > GD3 approximate to 2,6-isoGM3 approximate to GM4 approximate to GM1 sphingomyelin (SM). The extent of expansion reflected the strength of carbohydrate-carbohydrate interactions with Gg3, which were amplified by the clustering effect of carbohydrates in the glycoconjugate polystyrenes. The expansion of various proportions of GM3/SM mixed monolayers by PN(Gg3) was smaller than that of the GM3 monolayer, indicating the importance of clustered carbohydrates in the monolayer. The PN(Gg3)-induced expansion of the GM3 monolayer required no calcium ion, and the expansion was strongly inhibited in the presence of urea and N-acetamido sugars. These observations reveal a new aspect of the mechanism of the carbohydrate-carbohydrate interactions where the N-acetyl groups and hydrogen bonds between the carbohydrate moieties play important roles. The T-A isotherms using glycoconjugate polystyrenes have been demonstrated to be useful as a molecular probe for the carbohydrate -carbohydrate interactions.