A rheological study was performed on biofilms of mucoid Pseudomonas aeruginosa strains producing high molecular weight alginate as the major polysaccharide component of the extracellular polymeric substances (EPS). Environmental strain SG81 and clinical strain FRD1 produced O-acetylated alginate, and mutant FRD1153 derived from strain FRD1 was defective in alginate acetylation. Confluent biofilms were prepared by cultivating the bacteria at 36 °C for 24 h on membrane filters placed on Pseudomonas isolation agar. The rheological properties of these pure culture biofilms attached to the membrane filters were analysed using a rotating disc rheometer. The biofilms of all mucoid strains showed viscoelastic properties, with polymer (alginate) cross-linking caused predominantly by physical interactions in the form of entanglements. In dynamic frequency-sweep tests, we observed time-dependent plateau regimens, describing the dynamics of network structures. From these data, we could derive the concentration of elastically effective polymer chains as 5 (±1) x 1019 chains/l (limits are standard deviation) for 24 h old biofilms of P. aeruginosa SG81. Relaxation processes predicted an average lifetime of junction points of the order of 16 (±10) s. Pseudomona aeruginosa FRD1 revealed a more elastic polymer network when compared with the acetylation-defective strain FRD1153, which had a more viscous biofilm structure. With calcium (10 mmol/l) added to the growth medium, enhanced biofilm stability was observed, which was based mainly on Coulomb interactions between divalent calcium ions and the carboxylate groups of alginate, with entanglements making only minor contributions. The calcium-induced increase in polymer network stability was greater in biofilms of P. aeruginosa FRD1 than in the acetylation-defective strain FRD1153. These results indicate that acetyl groups as low molecular weight substituents of alginate polymers strongly influence the rheological behaviour of mucoid P. aeruginosa biofilms and are involved in the stabilization of polymer networks of the EPS matrix in both the absence and the presence of calcium ions.