The carbohydrate determinants of keratan sulphate recognized by three monoclonal antibodies (5-D-4, 1-B-4 and MZ15) have been investigated by solid-phase radioimmunoassay using bovine corneal keratan sulphate as the immobilized reference antigen. The antibodies appeared highly specific for sulphated poly(N-acetyllactosamine) sequences, for their binding was strongly inhibited by preparations of keratan sulphate, but not by glycoproteins with non-sulphated poly(N-acetyllactosamine) sequences of I and i antigen types, a desulphated keratan sulphate hexasaccharide, an array of neutral and sulphated mono- and disaccharides and other glycosaminoglycans. Inhibition of binding assays using a series of structurally characterized sulphated di, tetra-, hexa-, octa- and decasaccharides, and partially characterized larger oligosaccharides, isolated from bovine corneal keratan sulphate after digestion with endo-beta-galactosidase (see preceding two papers in this journal) showed that the smallest oligosaccharide reactive with all three antibodies was the linear pentasulphated hexasaccharide, E-II although antibody 1-B-4 reacted with a tetrasulphated analogue. The heptasulphated octasaccharide, G-III, was more active; among the structurally characterized keratan sulphate oligosaccharides the nonasulphated decasaccharide, I-IV, was the most active. Thus, the hepta- and octasaccharide sequences, indicated by brackets below are proposed as candidate antigenic structures recognized by the three monoclonal antibodies. (Formula: see text). Antibody 5-D-4 differs from the other two antibodies in reacting relatively strongly with a minor oligosaccharide which chromatographs as a hexasulphated octasaccharide, G-I, and most strongly with a minor sulphated, linear dodecasaccharide, J-II, which has been partially characterized [Tang, P.W., Scudder, P., Mehmet, H., Hounsell, E. F. & Feizi, T., unpublished results] and may contain N-sulphated glucosamine residues.