In order to identify amino acid residues in the Escherichia coli raffinose-H + permease (RafB) that play a role in sugar selection and transport, we first incubated E. coli HS4006 containing plasmid pRU600 (expresses inducible raffinose permease and α-galactosidase) on maltose MacConkey indicator plates overnight. Initially, all colonies were white, indicating no fermentation of maltose. Upon further incubation, 100 mutants red appeared. pRU600 DNA was prepared from 55 mutants. Five mutants transferred the phenotype for fermentation of maltose (red). Plasmid DNA from five maltose-positive phenotype transformants was prepared and sequenced, revealing three distinct types of mutations. Two mutants exhibited Val-35→Ala (MT1); one mutant had Ile-391→Ser (MT2); and two mutants had Ser-138→Asp, Ser-139→Leu, and Gly-389→Ala (MT3). Transport studies of [ 3 H]-maltose showed cells harboring MT1, MT2 and MT3 had greater uptake (P ≤ 0.05) than cells harboring wild-type RafB. However, [ 14 C]-raffinose uptake was reduced in all mutant cells (P ≤ 0.05) with MT1, MT2 and MT3 mutants compared to cells harboring wild-type RafB. Kinetic analysis showed enhanced apparent Km values for maltose and reduced Vmax/Km ratios for raffinose compared to wild-type values. The apparent Ki value of maltose for RafB indicates a competitive relationship between maltose and raffinose. Maltose "uphill" accumulation was greater for mutants (P ≤ 0.05) than for cells with wild-type RafB. Thus, we implicate residues in RafB that are responsible for raffinose transport and suggest that the substituted residues in RafB dictate structures that enhance transport of maltose.