Variations in biofilm formation by, and antibiotic resistance of, Pseudomonas aeruginosa PAO1 (wild type) and the quorum-sensing-deficient mutants PDO100 (Delta rhlI), JP1 (Delta lasI) and JP2 (Delta lasI Delta rhlI) were studied. For PAO1, the maximum-accumulation phase of biofilm formation began immediately and a plateau phase was reached after 24 h, whereas the quorum-sensing mutants showed 36-48 h lags before entering the maximum-accumulation phase. After 72 h, the cell density of the PAO1 biofilms was c. 0.8-1.2 log greater than for the mutants. On a unit protein basis, total polysaccharide production was similar for PAO1 and PDO100, whereas JP1 and JP2 biofilms accumulated only c. 36% of the PAO1 level after 72 h. Fluorescent micrographs revealed that the PAO1 biofilms were much thicker than those of the quorum-sensing-deficient mutants. In the case of the PAO1 and PDO100 biofilms, most cells were attached to the top of the biofilm layer, whereas the bottom layer consisted predominantly of polysaccharides. The JP1 and JP2 biofilms were closely packed with cells, and little polysaccharide was visible. Cells in PAO1 biofilms were little affected by kanamycin, even at 100 mg/L, whereas those in PDO100 biofilms were susceptible to the highest concentration of kanamycin (100 mg/L) but not to lower concentrations (10 and 50 mg/L). In contrast, cells in JP1 and JP2 biofilms were susceptible to kanamycin at all three concentrations.