Using an experimental in vitro culture system, we investigated the effect of clarithromycin on biofilm formation by a leucine-requiring Pseudomonas aeruginosa mutant strain (HU1). Biofilm formation on celldesks in a chemically defined medium was assessed by viable cell count as well as by measurement of glycocalyx production and scanning electron-microscopic observation. Cells proliferated exponentially until day 3 and remained stationary afterwards. The amount of glycocalyx, simultaneously semiquantified, showed a linear increase from day 1 to day 12. Scanning electron microscopy revealed firm biofilms on day 5. Three different concentrations of Clarithromycin (CAM) (minimum inhibitory concentration MIC 64 microg/ml) were added continuously at the early and late phases of biofilm formation, and the antibiofilm effect of CAM was evaluated by the changes in cell count and glycocalyx production. CAM was effective on biofilms at 100 microg/ml but neither at 1 nor at 10 microg/ml. It is suggested that glycocalyx production started following bacterial multiplication and continued even after the cells had entered the stationary phase to form mature biofilms. No antibiofilm effect of CAM was observed at sub-MIC.