The phytohormone abscisic acid (ABA) regulates physiologically important developmental processes and stress responses. Previously, we reported on Arabidopsis (Arabidopsis thaliana) L. Heynh. ahg mutants, which are hypersensitive to ABA during germination and early growth. Among them, ABA-hypersensitive germination3 (ahg3) showed the strongest ABA hypersensitivity. In this study, we found that the AHG3 gene is identical to AtPP2CA, which encodes a protein phosphatase 2C (PP2C). Although AtPP2CA has been reported to be involved in the ABA response on the basis of results obtained by reverse-genetics approaches, its physiological relevance in the ABA response has not been clarified yet. We demonstrate in vitro and in vivo that the ahg3-1 missense mutation causes the loss of PP2C activity, providing concrete confirmation that this PP2C functions as a negative regulator in ABA signaling. Furthermore, we compared the effects of disruption mutations of eight structurally related PP2C genes of Arabidopsis, including ABI1, ABI2, HAB1, and HAB2, and found that the disruptant mutant of AHG3/AtPP2CA had the strongest ABA hypersensitivity during germination, but it did not display any significant phenotypes in adult plants. Northern-blot analysis clearly showed that AHG3/AtPP2CA is the most active among those PP2C genes in seeds. These results suggest that AHG3/AtPP2CA plays a major role among PP2Cs in the ABA response in seeds and that the functions of those PP2Cs overlap, but their unique tissue- or development-specific expression confers distinct and indispensable physiological functions in the ABA response.