A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been identified among a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. To determine the importance of this motif within the lactose permease of Escherichia coli, a total of 28 site-directed mutations at the conserved first, fifth, sixth, eighth, ninth, and tenth positions were analyzed. A dramatic inhibition of activity was observed with all bulky mutations at the first-position glycine. Based on these results, together with sequence comparisons within the superfamily, it seems likely that small side chain volume (and possibly high β-turn propensity) may he structurally important at this position. The acidic residue at the fifth position was also found to be very important for transport activity and even a conservative glutamate at this location exhibited marginal transport activity. In contrast, many substitutions at the eighth-position glycine, even those with a high side chain volume and/or low β-turn propensity, still retained high levels of transport activity. Similarly, none of the basic residues within the motif were essential for transport activity when replaced individually by nonbasic residues. However, certain substitutions at the basic residue sites as well as the eighth- position glycine were observed to have moderately reduced levels of active transport of lactose. Taken together, the results of this study confirm the importance of the conserved loop 2/3 motif in transport function. It is suggested that this motif may be important in promoting global conformational changes within the permease.