The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coil. The lactose carrier transports the α-galactoside sugar melibiose as well as the β-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside β-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacy structural genes of individual mutants: Tyr-26→Asp, Phe-27→Tyr, Phe-29→Leu, Asp-240→Val, Leu-321→Gln, and His-322→Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp- 240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.