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One of the mechanisms through which some strategy I plants respond to Fe‐deficiency is an enhanced acidification of the rhizosphere due to proton extrusion. It was previously demonstrated that under Fe‐deficiency, a strong increase in the H ^plus; ‐ATPase activity of plasma membrane (PM) vesicles isolated from cucumber roots occurred. This result was confirmed in the present work and supported by measurement of ATP‐dependent proton pumping in inside‐out plasma membrane vesicles. There was also an attempt to clarify the regulatory mechanism(s) which lead to the activation of the H ⁺ ‐ATPase under Fe‐deficiency conditions. Plasma membrane proteins from Fe‐deficient roots submitted to immunoblotting using polyclonal antibodies showed an increased level in the 100 kDa polypeptide. When the plasma membrane proteins were treated with trypsin a 90 kDa …