Biofilm-forming bacteria such as Staphylococcus, Haemophilus, and Pseudomonas species resist phagocytosis by host immune cells and the actions of antimicrobial agents. In susceptible individuals, such as patients with cystic fibrosis (CF) or diffuse panbronchiolitis (DPB), strains of Pseudomonas aeruginosa produce a number of virulence determinants that permit colonization and infection of the respiratory tract. P aeruginosa strains isolated from CF and DPB patients typically have a mucoid colony morphology. This is due to the overproduction of alginate, an exopolysaccharide capsule that is composed of D-mannuronic and L-guluronic acids. In addition, the P aeruginosa type IV pilus mediates cell surface translocation by a process known as twitching motility. Both alginate production and twitching motility contribute to the virulence of P aeruginosa, as does the formation of biofilms. Biofilms bind cells and organic and inorganic materials to each other, and to a variety of substrata. Their tightly formed structure reduces antimicrobial activity, promotes bacterial adhesion to lung epithelia, and prevents bacterial dehydration. Prior work has suggested that macrolides have therapeutic value in patients with DPB and CF. We hypothesized that the improved clinical status of these patients was due, in part, to macrolides inhibiting the production of P aeruginosa virulence determinants. Traditionally, macrolides have not been considered to exhibit antipseudomonal activity, as their mean inhibitory concentration (MIC) values for clinical and environmental strains of the microbe range from 50 to 550 microg/mL. In this study, we found that sub-MIC levels of clarithromycin substantially inhibited twitching motility. In addition, the incubation of biofilm-grown P aeruginosa with clarithromycin altered the structure and architecture of the biofilm. Investigating the potential nonribosomal effects of macrolides on opportunistic pathogens such as P aeruginosa and elucidating the molecular mechanisms that underlie the inhibition of twitching motility may lead to more effective treatments of pulmonary infections in patients with CF and DPB.