The phenomenon of postantibiotic effect (PAE) encompasses not only the effects of bacterial growth inhibition but also the suppression of virulence factors. We tentatively designated the latter effect the postantibiotic suppression effect (PASE). The flagella of Gram-negative bacteria are involved in the development of biofilms. We measured the PASE of erythromycin (ERY) and azithromycin (AZM) on the expression of flagellin in Pseudomonas aeruginosa and Proteus mirabilis. Flagellin preparations were subjected to sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis and the flagellin band was identified by N-terminal amino acid sequence analysis. We thus evaluated the flagellin by the intensity of the band. The mean durations of the PAE of ERY and AZM on bacterial growth were 0.9 and 2.0 h for P. mirabilis, and 0.6 and 2.7 h for P. aeruginosa, respectively. The PASE of these drugs on flagellin expression was also observed. The apparent PASEs of ERY and AZM on flagellin were up to 5 h for P. mirabilis and up to 6 h for P. aeruginosa after a single 0.5 x minimum inhibitory concentration (MIC) treatment for 5 h. Our results suggest that certain combinations of antibiotics may have prolonged suppressive effects on the expression of virulence factors in certain Gram-negative bacteria.