A peptide motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been conserved in a large group of evolutionarily related membrane proteins that transport small molecules across the membrane. Within the superfamily, this motif is located in two cytoplasmic loops that connect transmemhrane segments 2 and 3 and transmembrane segments 8 and 9. In a previous study concerning the loop 2-3 motif of the lactose permease (A. E. Jessen-Marshall, N.J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995), it was shown that the first- position glycine and the fifth-position aspartate are critical for transport activity since a variety of site-directed mutations greatly diminished the rate of transport. In the current study, a similar approach was used to investigate the functional significance of the conserved residues in the loop 8-9 motif. In the wild-type lactose permease, however, this motif has been evolutionarily modified so that the first-position glycine (an α-helix breaker) has been changed to proline (also a helix breaker); the fifth position has been changed to an asparagine; and one of the basic residues has been altered. In this investigation, we made a total of 28 single and 7 double mutants within the loop 8-9 motif to explore the functional importance of this loop. With regard to transport activity, amino acid substitutions within the loop 8-9 motif tend to be fairly well tolerated. Most substitutions produced permeases with normal or mildly defective transport activities. However, three substitutions at the first position (i.e., position 280) resulted in defective lactose transport. Kinetic analysis of position 280 mutants indicated that the defect decreased the V(max) for lactose uptake. Besides substitutions at position 280, a Gly-288-to-Thr mutant had the interesting property that the kinetic parameters for lactose uptake were normal yet the rates of lactose efflux and exchange were approximately 10-fold faster than wild-type rates. The results of this study suggest that loop 8-9 may facilitate conformational changes that translocate lactose.