Porins mediate the diffusion of hydrophilic solutes across the outer membrane of mycobacteria, but the efficiency of this pathway is very low compared to Gram-negative bacteria. To examine the importance of porins in slow-growing mycobacteria, the major porin MspA of Mycobacterium smegmatis was expressed in Mycobacterium tuberculosis and Mycobacterium bovis. Approximately 20 and 35 MspA molecules per μm2 cell wall were observed in M. tuberculosis and M. bovis BCG, respectively, by electron microscopy and quantitative immunoblot experiments. Surface accessibility of MspA in M. tuberculosis was demonstrated by flow cytometry. Glucose uptake was twofold faster, indicating that the outer membrane permeability of M. bovis BCG to small and hydrophilic solutes was increased by MspA. This significantly accelerated the growth of M. bovis BCG, identifying very slow nutrient uptake as one of the determinants of slow growth in mycobacteria. The susceptibility of both M. bovis BCG and M. tuberculosis to zwitterionic β-lactam antibiotics was substantially enhanced by MspA, decreasing the minimal inhibitory concentration up to 16-fold. Furthermore, M. tuberculosis became significantly more susceptible to isoniazid, ethambutol and streptomycin. Fluorescence with the nucleic acid binding dye SYTO 9 was 10-fold increased upon expression of mspA. These results indicated that MspA not only enhanced the efficiency of the porin pathway, but also that of pathways mediating access to large and/or hydrophobic agents. This study provides the first experimental evidence that porins are important for drug susceptibility of M. tuberculosis. © 2004 SGM.