The phytohormone abscisic acid (ABA) inhibits blue light-induced apoplastic acidification of guard cells. The signal transduction pathway of ABA, mediating this response, was studied using ABA-insensitive (abi) mutants of Arabidopsis thaliana. Apoplastic acidification was monitored with a flat tipped pH-electrode placed on epidermal strips, in which only guard cells were viable. Blue light-induced apoplastic acidification was reduced by vanadate and diethylstilbestrol (DES), indicating involvement of plasma membrane-bound H+-ATPases. In wild type epidermal strips, ABA reduced blue light-induced acidification to 63%. The inhibition did not result from an increased cytoplasmic free Ca2+ concentration in guard cells, since factors that increase the Ca2+ concentration stimulated apoplastic acidification. Apoplastic acidification was not inhibited by ABA in abi1 and abi2 mutants. In abi1 epidermal strips ABA had no effect on the acidification rate, while it stimulated apoplastic acidification in abi2. The ABA response in both mutants could be partially restored with protein kinase and phosphatase inhibitors. The abi1 guard cells became ABA responsive in the presence of okadaic acid, a protein phosphatase inhibitor. In abi2 guard cells the wild type ABA response was partially restored by K-252a, a protein kinase inhibitor. Apoplastic inhibition is thus mediated through the protein phosphatases encoded by ABI1 and ABI2. The results with protein kinase and protein phosphatase inhibitors indicate that ABI1 and ABI2 are involved in separate signal transduction pathways.