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The regulatory role of intracellular pH changes and of transmembrane Cl− transport in the activation of Nicotiana tabacum L. pollen grains at a stage preceding in vitro germination was studied. The acidification of the cytosol with propionic acid hindered the germination of pollen grains, whereas its alkalization by fusicoccin-stimulated H+-ATPase activity of plasma membranes sharply increased the germination frequency with respect to control values. The activation of pollen grains was accompanied by the Cl− efflux. The blockage of Cl− efflux with 1 mM ethacrynic acid significantly decreased the intracellular pH and fully inhibited germination. The results allow assumption that the intracellular pH rise and Cl− efflux are prerequisites for pollen grain activation.