The I- and i-antigen activities of chemically synthesized, linear oligosaccharides of the neolacto series containing one, two or three N-acetyllactosamine (Gal beta 1----4GlcNAc) units have been tested by inhibition of binding of five anti-i and eight anti-I monoclonal antibodies to radioiodinated I- and i-active glycoproteins. The inhibitory activities of the milk oligosaccharides lacto-N-neotetraose (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc) and lacto-N-tetraose (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc) have also been determined. The results clearly show that: (a) the determinants that best fit the combining sites of anti-i antibodies are at least hexasaccharides of the neolacto series, (b) linear tetra- and hexasaccharides of the neolacto series can strongly inhibit the binding of anti-I antibodies of group 2 which are known to be primarily directed at the repeating Gal beta 1----4GlcNAc beta 1----3 domains of branched neolacto sequences, (c) the beta- but not the alpha-methyl anomer of the glycoside Gal beta 1----4GlcNAc beta 1-O-Me inhibits the binding of anti-I antibodies of group 1 which recognise the branch point sequence Gal beta 1----4GlcNAc beta 1----6-, (d) the reactivity of the beta-methylglycoside is impaired if the sequence is further elongated as in Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-O-Me, and (e) lacto-N-tetraose has no inhibitory activity with any of the anti-i or anti-I antibodies tested.