Solution culture studies have demonstrated that apoplasmic iron (Fe) deposited in the roots of dicotyledonous and graminaceous plants can be mobilized to improve plant iron status in conditions of limited Fe supply. The present study investigated the formation of the apoplasmic Fe pool in dicotyledonous (soybean and cucumber) and graminaceous (wheat) plants in a pot experiment. The pots had three compartments such that plants could take up Fe and other nutrients from two calcareous soils treated with different Fe forms without their roots touching with soil directly. In this way overestimating Fe accumulation in root apoplasm was avoided. The results showed that while the root d.wt of wheat did not vary when soils were supplied with different Fe resources, the root d. wt of soybean and cucumber supplied with FeEDTA decreased compared with the control (without Fe treatment). Supplying FeEDTA in the side compartment increased shoot d. wt and Fe concentration in shoots of all species. However, supplying Fe(OH)3 had no effect on shoot d. wt or Fe concentration in the shoots of any species. Soybean and cucumber accumulated little or no Fe in the root apoplasm in controls or in Fe(OH)3 treatments. By contrast, a large amount of Fe was deposited in the root apoplasm of wheat grown in similar conditions. Remarkably, when FeEDTA was supplied in the soils, large apoplasmic iron pools were formed in the roots of all three species. Therefore, in dicotyledonous plants grown on calcareous soils, little or no apoplasmic iron pool forms, because there is not enough available Fe in the soil solution and the plants have little ability to mobilize Fe3+ in the soil. By contrast, a larger apoplasmic iron pool could form in graminaceous plants at lower concentrations of available soil-Fe possibly by enhancing the release of phytosiderophores which could mobilize Fe3+ in the soil and then transfer the Fe3+-complexes to the root apoplasm.