Extracellular DNA is one of the major matrix components in Pseudomonas aeruginosa biofilms. It functions as an intercellular connector and plays a role in stabilization of the biofilms. Evidence that DNA release in P. aeruginosa PAO1 biofilms is controlled by the las-rhl and pqs quorum-sensing systems has been previously presented. This paper provides evidence that DNA release in P. aeruginosa PAO1 biofilms is also under iron regulation. Experiments involving cultivation of P. aeruginosa in microtitre trays suggested that pqs expression, DNA release and biofilm formation were favoured in media with low iron concentrations (5 mu M FeCIA and decreased with increasing iron concentrations. Experiments involving cultivation of P. aeruginosa in a flow-chamber system suggested that a high level of iron (1100 mu M FeCl3) in the medium suppressed DNA release, structural biofilm development, and the development of subpopulations with increased tolerance toward antimicrobial compounds. Experiments with P. aeruginosa strains harbouring fluorescent reporters suggested that expression of the pqs operon was induced in particular subpopulations of the biofilm cells under low-iron conditions (1 mu M FeCl3) but repressed in the biofilm cells under high-iron conditions (100 mu M FeCl3).