Mucoid strains of Pseudomonas aeruginosa overproduce the exopolysaccharide alginate, which is substituted with O-acetyl groups. Under nongrowing conditions in phosphate buffer, a mucoid clin. strain formed microcolonies on steel surfaces, while an acetylation-defective mutant was unable to form cell clusters. Enzymic degrdn. of alginate by alginate lyase prevented microcolony formation of the mucoid parent strain. In a continuous culture flow-cell system, using gluconate minimal medium, the mucoid strain with acetylated alginate formed microcolonies and grew into heterogeneous biofilms, whereas the acetylation-defective mutant produced a thinner and more homogeneous biofilm. A lowered viscosity of extracellular material from the acetylation-defective mutant indicated a weakening of exopolymer interactions by loss of acetyl groups. These results suggest that acetyl substituents are necessary for the function of high-mol.-mass alginate to mediate cell aggregation into microcolonies in the early stages of biofilm development by mucoid P. aeruginosa, thereby detg. the architecture of the mature biofilm.