Abscisic acid (ABA) is a plant hormone involved in seed development and responses to various environmental stresses. Oxidation of abscisic aldehyde is the last step of ABA biosynthesis and is catalysed by aldehyde oxidase (EC 188.8.131.52). We have reported the occurrence of three isoforms of aldehyde oxidase, AOalpha, AObeta and AOgamma, in Arabidopsis thaliana seedlings, but none oxidized abscisic aldehyde. Here we report a new isoform, AOdelta, found in rosette leaf extracts, which efficiently oxidizes abscisic aldehyde. AO delta was specifically recognized by antibodies raised against a recombinant peptide encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3 and AAO4). Functionally expressed AAO3 protein in the yeast Pichia pastoris showed a substrate preference very similar to that of rosette AOdelta. These results indicate that AOdelta is encoded by AAO3. AOdelta produced in P. pastoris exhibited a very low Km value for abscisic aldehyde (0.51 microM), and the oxidation product was determined by gas chromatography-mass spectrometry to be ABA. Northern analysis showed that AAO3 mRNA is highly expressed in rosette leaves. When the rosette leaves were detached and exposed to dehydration, AAO3 mRNA expression increased rapidly within 3 h of the treatment. These results suggest that AOdelta, the AAO3 gene product, acts as an abscisic aldehyde oxidase in Arabidopsis rosette leaves.