A simple method was used for testing the antibiotic susceptibility of adherent bacteria to plastic surfaces. Pseudomonas aeruginosa, Serratia marcescens, and Proteus mirabilis cells adhering to the bottom of a plastic tissue culture plate were incubated in serially diluted antibiotic solutions. After 24-h incubation the solutions were removed and a fresh medium without antibiotics was added to each well. The viability of the cells was judged by their growth after a further 24-h incubation. In our assay system, we employed a short incubation time (1-h) involving adherence of bacteria to a surface for the purpose of minimizing the effect of the glycocalyx on antibiotic activity. Even if the bacteria did not form a biofilm, the minimal bactericidal concentrations for adherent bacteria (MBCADs) markedly elevated. The MBCADs of ofloxacin well correlated with the bacteriological eradication by ofloxacin treatment for urinary tract infections (UTIs) associated with indwelling urinary catheters, whereas the minimal inhibitory concentrations did not show a correlation. Kinetic studies showed that adherent Pseudomonas aeruginosa had a 2 h-lag time before logarithmically growing when these bacteria were incubated in Mueller-Hinton broth without antibiotics. The tolerance demonstrated by adherent cells is likely to play a role in the difficulties encountered in the antimicrobial chemotherapy of biofilm infections. Moreover, our assay system was considered to be useful in the therapeutic selection of antibiotics for these infections.