The growth rate of biofilm cells of a leucine-requiring mutant Pseudomonas aeruginosa HU1 was regulated by the leucine concentration in the chemically defined medium, and the effect of the growth rate of biofilm cells on the antimicrobial activities of the antimicrobial agents piperacillin (PIPC), imi-penem (IPM) and ofloxacin (OFLX) were evaluated. PIPC showed little effect on the biofilm bacteria regardless of the leucine concentration in the medium. IPM showed weak bactericidal activity to biofilm cells; activity was greater in younger biofilm cells growing in high concentrations of leucine. On the other hand, OFLX revealed strong bactericidal activity to biofilm bacteria regardless of the growth rate. Our data suggest that the bactericidal action of antimicrobial agents to biofilm bacteria is different from that to planktonic bacteria. The bioassay using mutants with regulated growth is useful for the evaluation of the efficacy of antimicrobial agents against biofilm bacteria.