Rapid filtration assays were used to characterize glutamate/cation uptake in brush-border membrane vesicles from the larval midgut of the lepidopteran Manduca sexta. At pH 10.5, which is close to the physiological pH in the midgut of M. sexta, an inwardly directed K+ gradient stimulated glutamate uptake, suggesting that glutamate was symported. Gradients of Na+ or Li+ were less effective. Neither Rb+ nor Cs+ stimulated glutamate uptake. Anion-specificity was less pronounced: the accumulation maximum was only slightly higher with thiocyanate (SCN-) than with Cl-, although initial uptake was noticeably faster with thiocyanate. A distinct set of amino acids that would cis-inhibit or trans-elicit glutamate uptake was not found. Even L-glutamate itself did not elicit accumulations of labeled glutamate. Taken together, these results suggest that a glutamate-specific symporter may not be present. Moreover, because glutamate symport was found to be electroneutral in vitro whereas amino acid uptake is electrophoretic in vivo, we infer that symport with K+ may not be an important mechanism of glutamate translocation by M. sexta midgut.