For the first time, phytosiderophore (PS) release of wheat (Triticum aestivum cv Tamaro) grown on a calcareous soil was repeatedly and nondestructively sampled using rhizoboxes combined with a recently developed root exudate collecting tool. As in nutrient solution culture, we observed a distinct diurnal release rhythm; however, the measured PS efflux was c. 50 times lower than PS exudation from the same cultivar grown in zero iron (Fe)-hydroponic culture.Phytosiderophore rhizosphere soil solution concentrations and PS release of the Tamaro cultivar were soil-dependent, suggesting complex interactions of soil characteristics (salinity, trace metal availability) and the physiological status of the plant and the related regulation (amount and timing) of PS release.Our results demonstrate that carbon and energy investment into Fe acquisition under natural growth conditions is significantly smaller than previously derived from zero Fe-hydroponic studies. Based on experimental data, we calculated that during the investigated period (21–47 d after germination), PS release initially exceeded Fe plant uptake 10-fold, but significantly declined after c. 5 wk after germination.Phytosiderophore exudation observed under natural growth conditions is a prerequisite for a more accurate and realistic assessment of Fe mobilization processes in the rhizosphere using both experimental and modeling approaches.