The classical action of the hormone 1,25-dihydroxyvitamin D3 (VD) is the regulation of calcium metabolism. In contrast, the peptide hormone atrial natriuretic factor (ANF) is one of the few known nonclassical VD responding genes. We screened the promoter of the rat ANF gene and identified a typical VD receptor (VDR) binding site formed by a direct repeat of two hexameric core binding motifs spaced by three nucleotides, between positions -907 and -891. Like most of the DR3-type VD response elements this sequence is bound with high affinity (Kd = 0.53 nM) by a heterodimer formed by VDR and retinoid X receptor. In a heterologous promoter context one copy of this sequence mediated an about fourfold gene activation by VD and a half-maximal activation (EC50) value of 0.48 nM VD. This characterizes the identified sequence as one of the most potent VD response elements.