Three rhamnose-binding lectins were purified from the roe of Osmerus eperlanus mordax (olive rainbow smelt) by affinity chromatography and ion-exchange chromatography. The apparent molecular weights of Osmerus eperlanus mordax lectin (OML) -1, -2 and -3 were 25000, 32000 and 26000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. On native PAGE, these three lectins showed different migration patterns (Rm value; 0.37, 0.53 and 0.66, respectively). OMLs agglutinated rabbit and human type B erythrocytes and sarcoma 180 cells, but not human type A and 0 erythrocytes and AH109A cells. The most effective monosaccharide inhibitor was L-rhamnose. L-Mannose and D-galactose were also good inhibitors. Furthermore, OML-induced hemagglutination was inhibited more strongly by melibiose or raffinose rather than lactose or lactulose. Therefore, OMLs are L-rhamnose/alpha-D-galactosyl type lectins. OMLs did not require a detergent, when extracted from crude material, and Ca2+, Mg2+, EDTA and dithiothreitol were not necessary for the OML-induced hemagglutination activities. The OMLs had similar N-terminal amino acid sequences.