Deletion of 5 residues (Delta5) from the central cytoplasmic loop of the lactose permease of Escherichia coli has no significant effect on expression or activity, whereas Delta12 leads to increased rates of permease turnover after membrane insertion and decreased transport activity, and Delta20 abolishes insertion and activity. By expressing Delta12 or Delta20 in two halves, both expression and activity are restored to levels approximating wild type. Replacing deleted residues with random hydrophilic amino acids also leads to full recovery. However, introduction of hydrophobic residues decreases expression and activity in a context-dependent manner. Thus, a minimum length of the central cytoplasmic loop is vital for proper insertion, stability, and efficient transport activity, because of constraints at the cytoplasmic ends of helices VI and VII. Furthermore, the results are consistent with the idea that the middle cytoplasmic loop provides a temporal delay between insertion of the first six helices into the membrane before insertion of the second six helices.