Isolated ciliary axonemes from pig trachea were exposed to increasing concentrations of purified Pseudomonas aeruginosa rhamnolipid. This is a defined ciliary system allowing observation of direct impairment of functional axonemes. Axonemal motility and ATPase activity were decreased in proportion to rhamnolipid concentrations. ATPase-associated proteins observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dynein arms seen in ultra-structural cross sections progressively disappeared from axonemes with exposure to rhamnolipid. These four independent measures establish that the rhamnolipid removes the ATPase-containing outer dynein arms from the ciliary axoneme, thereby rendering the axoneme immotile.