Factors that regulate and induce stalk formation by the iron-oxidizing and stalk-forming bacterium Gallionella ferruginea were studied in laboratory cultures and in situ. A stalk-forming strain, Sta(+), and a non-stalk-forming strain, Sta(-), were used for comparative studies of the benefits associated with the stalk. Two different growth media were used: a ferrous sulfide medium (FS-medium), with slow oxidation of iron giving high concentrations of toxic oxygen radicals and a ferrous carbonate medium (FC-medium), with fast iron oxidation giving low concentration of the toxic oxygen radicals. It was found that Sta(+) cells grown in the FS-medium survived 3 weeks longer than Sta(-) cells grown in the FS-medium. When each strain was grown in the FC-medium, the Sta(-) cells had an advantage and survived 8 weeks longer than the Sta(+) cells. No difference in survival was found for Sta(+) cells grown in FS-medium compared to growth in FC-medium. In laboratory cultures, the average stalk length per cell values were 7-2.5 times higher (92 h and 150-300 h growth, respectively) in a medium with 620 μM iron than in a medium with 290 μM iron. Gallionella ferruginea Sta(+) outcompeted Sta(-) cells when inoculated as mixed populations in FC-medium. It has previously been suggested that stalk formation in vitro is induced by oxygen. To confirm this observation, biofilm development in natural waters was studied in two wells, one with trace amounts of oxygen (LH) and one without (TH). A dense biofilm developed on surfaces exposed to flowing well LH water, but no biofilm developed in well TH. Stalks were formed in water samples from both wells when allowed to make contact with air. This work demonstrates for the first time that the stalk has a protecting function against the toxic oxygen radicals formed during the chemical iron oxidation. It also shows that it is the oxidation rate of the ferrous iron and not its concentration that is harmful to the cells. The stalk gives G. ferruginea a unique possibility to colonize and survive in habitats with high contents of iron, inaccessible for bacteria without a defense system against the oxidation of iron.