The *ABI3* gene, which is involved in seed development and germination in response to abscisic acid (ABA), was isolated by positional cloning in Arabidopsis. A detailed RFLP map of the *abi3* region was constructed, and an RFLP marker closely linked to the *abi3* locus was identified. By analyzing overlapping cosmid clones containing this marker, the *abi3* locus was localized within a 35-kb region. An 11-kb subfragment was shown to complement the mutant phenotype in transgenic plants, further refining the locus's position. The candidate *ABI3* gene was identified within this fragment and was found to be expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. The most severe *abi3-4* allele showed a 40% reduction in protein size due to a premature stop codon introduced by a point mutation. The predicted *ABI3* protein displays high similarity to the maize *viviparous-1* (VP1) protein in several regions, particularly in a region encoded by four conserved exons. This study demonstrates the feasibility of isolating an Arabidopsis locus by positional cloning and highlights the potential role of the *ABI3* protein in ABA-regulated gene expression during seed development.The *ABI3* gene, which is involved in seed development and germination in response to abscisic acid (ABA), was isolated by positional cloning in Arabidopsis. A detailed RFLP map of the *abi3* region was constructed, and an RFLP marker closely linked to the *abi3* locus was identified. By analyzing overlapping cosmid clones containing this marker, the *abi3* locus was localized within a 35-kb region. An 11-kb subfragment was shown to complement the mutant phenotype in transgenic plants, further refining the locus's position. The candidate *ABI3* gene was identified within this fragment and was found to be expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. The most severe *abi3-4* allele showed a 40% reduction in protein size due to a premature stop codon introduced by a point mutation. The predicted *ABI3* protein displays high similarity to the maize *viviparous-1* (VP1) protein in several regions, particularly in a region encoded by four conserved exons. This study demonstrates the feasibility of isolating an Arabidopsis locus by positional cloning and highlights the potential role of the *ABI3* protein in ABA-regulated gene expression during seed development.