A superfamily of transport proteins, which includes the lactose permease of Escherichia coli, contains a highly conserved motif, G-X-X-X-D/E-R/K-X-G- R/K-R/K, in the loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. Previous analysis of this motif in the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995) has shown that the conserved glycine residue found at the first position in the motif (i.e., Gly-64) is important for transport function. Every substitution at this site, with the exception of alanine, greatly diminished lactose transport activity. In this study, three mutants in which glycine-64 was changed to cysteine, serine, and valine were used as parental strains to isolate 64 independent suppressor mutations that restored transport function. Of these 64 isolates, 39 were first-site revertants to glycine or alanine, while 25 were second-site mutations that restored transport activity yet retained a cysteine, serine, or valine at position 64. The second-site mutations were found to be located at several sites within the lactose permease (Pro-28 → Ser, Leu, or Thr; Phe.29 → Ser; Ala-50 → Thr, Cys-154 → Gly; Cys-234 → Phe; Gin-241 → Leu; Phe-261 → Val; Thr-266 → Iso; Val-367 → Glu; and Ala-369 → Pro). A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains. The apparent K(m) values of the Cys-64, Ser-64, and Val-64 parental strains were 0.8 mM, 0.7 mM, and 4.6 mM, respectively, which was similar to the apparent K(m) of the wild-type permease (1.4 mM). In contrast, the V(max) values of the Cys-64, Ser-64, and Val-64 strains were sharply reduced (3.9, 10.1, and 13.2 nmol of lactose/min · mg of protein, respectively) compared with the wild-type strain (676 nmol of lactose/min · mg of protein). The primary effect of the second-site suppressor mutations was to restore the maximal rate of lactose transport to levels that were similar to the wild-type strains. Taken together, these results support the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane. According to our tertiary model, this site is at an interface between the two halves of the protein.