Recently, it has been shown that trehalose and mycolic acids are essential for the growth of Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium smegmatis, and important but not indispensable to the survival of Corynebacterium glutamicum. Therefore, to investigate the function of mycolic acids in both the permeability of the cell wall to small nutrients and antibiotics, and the excretion of amino acids by C. glutamicum, a trehalose-deficient mutant of the L-lysine producer ATCC 21527, designated LP Delta treS Delta otsA Delta treY, was constructed. By using different carbon sources in either the presence or the absence of external trehalose, a set of endogenously trehalose-free LP Delta treS Delta otsA Delta treY cells that exhibited various mycolate contents was generated. The results showed that the structure of the arabinogalactan of these different cell types of LP Delta treS Delta otsA Delta treY was not affected when the mycolic acid layer was either missing or impaired. Nevertheless, cells were more susceptible to antibiotics, and the permeability of their cell walls to glycerol was increased. Interestingly, a concomitant increase in the excretion of both L-lysine and L-glutamate was also observed, indicating that the mycolic acid content of the permeability barrier (and not only the peptidoglycan and/or the arabinogalactan) is implicated in the glutamate excretion process.