Using the rapid filtration technique, cation gradient driven leucine, alanine and phenylalanine uptake by brush-border membrane vesicles (BBMV) from the highly studied model insect, Manduca sexta, is characterized at the physiological pH of 10. The vesicles are sealed and nonspecific binding is small. Almost identical initial time courses of leucine uptake are obtained whether the vesicles are osmotically balanced initially or at equilibrium. The maximum accumulation values are also similar and the equilibrium values are identical with either treatment. Equilibrium is reached by 60 min. Amino acid accumulation is cation gradient dependent and is abolished by 18 microM valinomycin. Uptake of all three amino acids occurs over a broad pH range with maximum rates at approximately pH 10 and lower rates at pH 7.5. The cation selectivity of phenylalanine and alanine uptake changes with pH; the sequence is K+ > Na+ > Cs+ >> Rb+ = Li+ at pH 10.0, whereas K+ = Na+ at pH 8.0; the selectivity of leucine uptake is K+ = Na+ > Cs+ >> Rb+ = Li+ at pH 10. Maximum K+ driven accumulation of all three amino acids decreases with anions in the order: SCN- > NO3- > Cl- = CO(3)2- = So(4)2- = HPO(4)2- > gluconate-.Vmax values are similar for all three amino acids. There are large differences in initial uptake rates (leucine > phenylalanine = alanine), and maximum accumulation values (leucine > phenylalanine > alanine).