A lithium ion-selective electrode was constructed using N,N‘-diheptyl-N,N‘-5,5-tetramethyl-3,7-dioxanonandiamid as a Li+ ionophore. Lithium ion-sugar cotransport via the melibiose transport system was measured with this electrode. Influx of methyl-beta-D-thiogalactoside, methyl-alpha-D-galactoside, methyl-beta-D-galactoside, and D-galactose elicited uptake of Li+. This Li+ uptake was not observed when the melibiose carrier was not present in the cells or the carrier was inactivated. Melibiose caused a small amount of Li+ uptake, indicating that Li+-melibiose cotransport proceeds inefficiently. Raffinose, another substrate, did not cause detectable Li+ transport. In mutant cells which showed altered cation coupling (Niiya, S., Yamasaki, K., Wilson, T. H., and Tsuchiya, T. (1982) J. Biol. Chem. 257, 8902-8906), Li+-melibiose cotransport was clearly demonstrated. Alteration in substrate specificity was also shown in the mutants.