P. aeruginosa isolates carrying a mutation in Domain V of the 23S rRNA gene showed a resistant profile in liquid and solid Mueller-Hinton and RPMI-1640 medium. Domain V of the 23S rRNA gene was sequenced in 55 P. aeruginosa isolates and analyzed for known mutations conferring resistance to macrolides (A2045T, A2045G, A2046T, A2046G and C2598T). Isolates with a wild-type Domain V of the 23S rRNA gene were split into two subgroups according to their resistance profile on RPMI-1640 agar. Azithromycin susceptibility was measured by disk diffusion (a); and broth microdilution (b); in Mueller-Hinton (circles) and RPMI-1640 (triangles) medium. Data show the median + interquartile range, Nonparametric Mann-Whitney Test, *** <0.0001. Disk size: 6 mm, 15 µg azithromycin.