In this study, we evaluated the permeation of piperacillin (PIPC), imipenem (IPM), amikacin (AKM), gentamicin (GM), ofloxacin (OFLX), levofloxacin (LVFX), ciprofloxacin (CPFX) and sparfloxacin (SPFX) through Pseudomonas aeruginosa biofilm with a simple new method. Bacteria used were a leucine-requiring mucoid mutant. Bacteria were grown on the membrane of a cell culture insert in chemically defined medium and incubated at 37 degrees C for 5 days. At days 0, 1, 3 and 5, the penetration rates through the biofilms were measured. PIPC and IPM demonstrated relatively high permeation both with penetration rates at day 5 of 50%, whereas AMK and GM, which are aminoglycosides, showed low permeation both with penetration rates after day 1 of less than 25%. Among the 4 fluoroquinolones, LVFX and SPFX demonstrated excellent permeation with penetration rates that reached 100% from day 0 to 5, while OFLX and CPFX showed almost the same permeation as IPM. This method of measuring penetration rates of antimicrobial agents through biofilm is very simple and useful for the evaluation of antibiotics against biofilm-forming bacteria.