Pseudomonad proteases disrupted the function and structure of demembranated cilia (axonemes) extracted from porcine tracheae. Proteolytic degradation by the two pseudomonad proteases elastase and alkaline protease and by trypsin and subtilisin impaired motility of ATP-activated axonemes. In addition, electron microscopic observation of negatively stained axonemes indicated that exposure to proteases caused dissociation into individual doublet or singlet microtubules. Inhibition of motility and axonemal fraying occurred when axonemes were treated with <5 U of proteolytic activity of any of the four proteases tested. When the effects of 2 U of each protease were compared, trypsin and subtilisin were able to produce immotility in less time than pseudomonad elastase and alkaline protease, while alkaline protease and subtilisin caused the most axonemal fraying in 10 min. Proteolytic digestion of axonemal proteins was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All four proteases cleaved dynein proteins (proteins necessary for motility), though treatment with trypsin resulted in the most extensive solubilization of axonemal proteins. Trypsin and subtilisin both produced more changes in the protein profiles of treated axonemes, using fewer units of proteolytic activity, than the pseudomonad proteases. However, the limited alteration of only a few axonemal proteins by pseudomonad proteases indicates that cleavage need not be extensive to product dysfunction. Thus, ciliary axonemes are susceptible to proteolytic attack. Degradation of axonemal proteins by pseudomonad proteases, which are released during active infection, may contribute to the impaired ciliary function associated with pseudomonad colonization of the respiratory tract.