The streptomycin-treated mouse colonization model was used to investigate the role of the Fe2+ uptake system (Feo) of Escherichia coli K12 in the colonization of the mouse intestine. Mutants impaired in the uptake of Fe2+ ions were shown to be deficient also in their colonization ability. Both enterochelin-producing and enterochelin-nonproducing Escherichia coli feo mutants were unable to colonize the mouse intestine. These results demonstrated that Fe(II) is an essential source of iron for E. coli grown in the intestine.