Lactose permease mutants, which were previously isolated in sugar specificity studies, were screened for their abilities to transport the trisaccharide maltotriose. Six multiple mutants (e.g., five double mutants and one triple mutant) were identified as forming fermentation-positive colonies on maltotriose MacConkey plates and were also shown to grow on maltotriose minimal plates. All of these multiple mutants contained a combination of two or three amino acid substitutions at position 177, 236, 306, or 322 within the permease. In contrast, none of the corresponding single mutants at these locations were observed to exhibit an enhanced rate of maltotriose transport. In whole-cell assays, the multiple mutants were shown to transport relatively long α-nitrophenylglucoside (αNPG) molecules. In certain cases, αNPG molecules containing up to four glucose residues in addition to the nitrophenyl group were shown to be transported to a significant degree. Overall, the abilities of lactose permease mutants to transport maltotriose and long αNPGs are discussed with regard to the dimensions of the sugar and the mechanism of sugar transport.