The binding of O-methyl and fluorodeoxy derivatives of methyl β-lactoside to the Ricinus communis toxin (RCA60) and agglutinin (RCA120) was studied in order to determine the donor/acceptor relationships of the hydrogen bonds between the hydroxyl groups of methyl β-lactoside and the binding sites of the lectins. Free energy contributions of the hydrogen bonds at each position have been estimated from these data and from those previously reported for the monodeoxy derivatives [Rivera-Sagredo, A., Solís, D., Díaz-Mauriño, T., Jiménez-Barbero, J. & Martín-Lomas, M. (1991) Eur. J. Biochem. 197, 217–228; Rivera-Sagredo, A., Jiménez-Barbero, J., Martín-Lomas, M., Solís, D. & Díaz-Mauriño, T. (1992) Carbohydr. Res. 232, 207–226]. The nature of the groups of the lectins involved in hydrogen bonding has been predicted on the basis of the free energy data. Analysis of the results indicates that both the C-3′ and C-4′ hydroxyl groups act as hydrogen-bond donors to charged groups of both RCA60 and RCA120. The C-6′ and probably also the C-2′ hydroxyl groups participate both as donors and as acceptors of two hydrogen bonds with neutral groups of the lectins. And finally, the C-6 hydroxyl group possibly acts as a donor of a weak hydrogen bond to a neutral group in RCA60, but not in RCA120. The results provide a molecular basis to explain some features of the binding specificity of the lectins. Comparison of RCA60 binding data with the recently refined X-ray crystal structure of the RCA60-lactose complex shows similarities but also some discrepancies that can be attributed to the marked influence of the pH on the carbohydrate—lectin interaction.