1,25-dihydroxyvitamin D, through association with its cognate nuclear receptor, has been shown to have important effects in the cardiovascular and renal systems. We have shown previously that the liganded vitamin D receptor (VDR) inhibits hypertrophy and expression of hypertrophy-sensitive genes (i.e. those encoding atrial natriuretic peptide [ANP], brain natriuretic peptide and alpha skeletal actin) in neonatal cardiac myocytes. In the present study we confirm a time-, ligand- and retinoid X receptor-dependent, VDR-mediated suppression of human ANP gene promoter activity. Conventional deletion analysis demonstrated that the promoter region positioned between -217 and -104 is required for the VDR-dependent suppression of the hANP promoter. Mutation of two functional CArG elements, including one located within this critical region, failed to reverse the suppression. We found no evidence that the liganded VDR is capable of associating directly with regulatory elements positioned between -217 and -104. We conclude that the inhibition may arise from protein-protein interactions between the liganded VDR and stimulatory transcription factors that bind in this region.