Azithromycin differs chemically from erythromycin by having an extra positive charge created by the presence of a methyl-substituted nitrogen in the 15-membered macrolide ring. This results in substantially increased potency against Gram-negative bacteria. Therefore, the possibility was considered that azithromycin was taken across the outer membrane of Escherichia coli by the self-promoted uptake route, which is utilized by other cationic antibiotics including polymyxins and aminoglycosides. Azithromycin, like polymyxin B and gentamicin, demonstrated equal activity against porin-sufficient and porin-deficient E. coli strains but its MIC was increased eight-fold by magnesium supplementation. Nevertheless, an outer membrane-altered mutant DC2 was eight-fold more susceptible than its parent strain UB1005 to azithromycin, indicating that the outer membrane was a permeability barrier to this macrolide. A mutant SC9252 which had an alteration in the self-promoted uptake of polymyxin and gentamicin, was more resistant to azithromycin, polymyxin and gentamicin compared to its parent SC9251. Further azithromycin, like polymyxin B and gentamicin, was capable of weakly permeabilizing cells to the hydrophobic fluorophor 1-N-phenyl-naphthylamine, a process antagonized by Mg2+. The monobasic macrolide erythromycin on the other hand was less affected by the SC9252 mutation, less effectively antagonized by Mg2+, and was a far less effective permeabilizer than dibasic azithromycin. These data are consistent with the hypothesis that the improved efficacy of azithromycin compared to erythromycin against E. coli reflects its better access to the self-promoted uptake pathway due to its additional positive charge.