The relationship between the apoplastic pH in young sunflower roots (Helianthus annuus L.) and the plasmalemma ferric chelate reductase (FC-R; EC activity in roots was investigated. The hypothesis was tested that a high apoplastic pH depresses FC-R activity, thereby restricting the uptake of Fe2+ into the cytosol. Until recently, little has been known about this relationship, because pH and redox reaction measurements are difficult to perform within the confines of the root apoplast. We recorded the apoplastic pH by means of the fluorescence ratio in conjunction with video microscopy by covalently tagging fluorescein boronic acid to OH groups of the root cell wall. FeIII reduction was measured using a similar approach by tagging ferrozine diboronic acid with OH groups of the cell wall. Ferrozine forms an Fe2+ complex, thus indicating the reduction of ferric iron. In roots bathing in buffered outer solutions of different pH, a high pH sensitivity of apoplastic FeIII reduction was found, with the highest ferric iron reduction rates at an apoplastic pH of 4.9; above an apoplastic pH of 5.3, no reduction was observed. Nitrate in the bathing solution increased the apoplastic pH and hence depressed the FeIII reduction; ammonium had the reverse effect. Nitrate together with HCO3, a combination which is typical of calcareous soils, had the strongest depressing effect. From the results, it can be concluded that the main reason for the frequently occurring iron deficiency chlorosis of plants grown on calcareous soils is the inhibition of FeIII reduction in the apoplast, and hence Fe2+ uptake into the cytosol.