Although an APETALA2 (AP2)-type transcription factor, WRINKLED1 (WRI1), has been shown to be required for accumulation of triacylglycerols (TAGs) in Arabidopsis seeds, its direct target genes have not been established. Overexpression of WRI1 up-regulated a set of genes involved in fatty acid (FA) synthesis in plastids, including genes for a subunit of pyruvate kinase (Pl-PKbeta1), acetyl-CoA carboxylase (BCCP2), acyl carrier protein (ACP1), and ketoacyl-acyl carrier protein synthase (KAS1), while expression of these genes is reduced in mutants with reduced WRI1 expression. Transient expression of LUC reporter genes with the proximal sequences upstream from the ATG codon of Pl-PKbeta1, BCCP2, and KAS1 in protoplasts was activated by co-expression of WRI1, and recombinant WRI1 bound to these upstream sequences in vitro. The seven WRI1 binding sites shared a sequence [CnTnG](n)(7)[CG], where n is any nucleotide designated as the AW-box, and mutations in AW-boxes near the transcription start site and in the 5'-untranslated region of Pl-PKbeta1 abolished activation by WRI1 in protoplasts and expression during seed maturation. Although expression of genes for the synthesis of TAGs and packaging into oil bodies in the endoplasmic reticulum in developing seeds required WRI1, their expression was not up-regulated by WRI1 overexpression. Thus, WRI1 promotes the flow of carbon to oil during seed maturation by directly activating genes involved in FA synthesis and controlling genes for assembly and storage of TAG.