The Arabidopsis aldehyde oxidase 3 (AAO3) gene encodes an enzyme that catalyzes the final step of ABA biosynthesis. AAO3 has been shown to be the major AAO involved in ABA biosynthesis in leaves under stress conditions. On the other hand, less severe phenotypes of the aao3 seeds suggested that other AAO(s) might also be involved in ABA biosynthesis in seeds. Among four AAOs (AAO1-AAO4), AAO1 and AAO4 were the AAO expressed most abundantly in dry seeds and developing siliques, respectively. Unlike aao3, single loss-of-function mutants for AAO1 and AAO4 (aao1 and aao4), failed to show significant changes in endogenous ABA levels in seeds when compared with wild type. While aao3 seed germination was resistant to the gibberellin biosynthesis inhibitor, uniconazole, aao1 and aao4 showed no resistance and were similar to wild type. These results indicate that AAO3, but not AAO1 or AAO4, plays an important role in ABA biosynthesis in seeds. Mutations of AAO1 or AAO4 in the aao3 mutant background enhanced ABA deficiency in seeds, demonstrating that both gene products contribute partially to ABA biosynthesis in the aao3 mutant background. However, considering the enzymatic characters of AAO1 and AAO4, their involvement in ABA biosynthesis in wild-type seeds may be negligible. We have concluded that AAO3 is the AAO that plays a major role in ABA biosynthesis in Arabidopsis seeds as well as in leaves.