Pseudomonas aeruginosa is an increasingly prevalent opportunistic human pathogen. It secretes a variety of hydrolases, many of which contribute to virulence or are thought to play a role in nutrition of the bacterium. However, potential roles of these extracellular enzymes in biofilm formation or composition of the extracellular polymeric substances (EPS) have not been studied. The aim of this study was to compare biofilms of the mucoid P. aeruginosa strain SG81 and its derivatives overexpressing different extracellular hydrolases. For this the genes for lipases LipA and LipC, the esterase EstA and elastase LasB were moderately overexpressed from plasmids in strain SG81. Biofilms were grown in flow-cells, analyzed by confocal laser-scanning microscope (CLSM) and the effects on EPS composition and physicochemical properties were investigated. Whereas lipA overexpression revealed no significant differences, the other enzymes influenced biofilm structure and/or EPS properties. In contrast to the parent strain, cells overproducing LipC were more heterogeniously distributed in the biofilm which was predominently composed of microcolonies consisting of highly packed cells. However, LipC overproduction had no influence on the EPS composition. Overproduction of EstA led to differences in biofilm architecture and resulted in premature biofilm dissolution. Compared to the parent strain the EPS showed differences in the concentrations of carbohydrates and rhamnolipids. Moreover, hydrophobicity and viscosity of the EPS were significantly increased. Overproduction of LasB had the most pronounced effects on biofilms: a drastically reduced ability to form an elaborate biofilm architecture resulting in a monolayer of unevenly distributed cells even after prolonged times of growth. Furthermore, the concentration of alginate was reduced, whereas the total concentration of carbohydrates increased. Like in the EstA overproducing strain the hydrophobicity and viscosity of the EPS were increased. With Gfp-enzympromotor-fusions it was possible to located a continuous transcription of lipA in all cells of the biofilm. In Contrast the transcription activity of the estA gene occurred only in a few cells at a later state of biofilm developement. These results clearly indicate that overexpression of extracellular enzymes can affect EPS composition and properties, thereby influencing biofilm development. Altogether, this data provides strong evidence that extracellular enzymes, in addition to their roles in virulence and nutrition, have a key role in the differentiation of microbial biofilms.