Antibodies were made against the two-chain lectin Lath-O from the seeds of Lathyrus odoratus as well as its isolated light (alpha) and heavy (beta) chains. These antibodies were used to antigenically compare the Lath-O with other two-chain lectins like Lath-S, lentil and Vicia cracca glc specific and with one-chain lectins like Con A, PHA, peanut and soybean. Sensitive ELISA tests showed that there was a distinct antigenic cross-reaction between the one-chain and two-chain lectins both by using antibodies against Lath-O whole molecules and its isolated beta-chains, while the anti-Lath-O alpha-chain antibodies barely reacted at all even with Lath-O alpha-chains. The antibodies against Lath-O whole molecules also strongly inhibited the mitogenic responses induced by two-chain lectins. Antibodies reacting inside the carbohydrate-binding site of the lectins were isolated by eluting them with glucose from lectin-Sepharose columns and antibodies reacting outside the carbohydrate-binding site were subsequently eluted with guanidine or buffer with low pH. The two antibody fractions were separately used to antigenically compare one-chain and two-chain lectins, demonstrating that both kinds of antibodies showed cross-reaction between one-chain and two-chain lectins. Both the antibodies presumably reacting inside the carbohydrate-binding site of the lectins and those reacting outside the carbohydrate-binding site inhibited haemagglutination activity of two-chain lectins, while haemagglutination by one-chain lectins was unaffected. The carbohydrate-binding site specificity of the glucose-eluted antibodies was assessed by glucose-inhibition in ELISA tests. Furthermore, galactose neither inhibited in these tests nor eluted antibodies from lectin-Sepharose columns. Control experiments were also performed with normal rabbit IgG.