We have examined the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of the rat atrial natriuretic peptide (ANP) gene and the secretion of the encoded protein product in neonatal rat cardiac myocyte cultures. 1,25(OH)2D3 effected a dose- and time-dependent inhibition of agonist-stimulated ANP secretion, which was accompanied by a reduction in the levels of the ANP mRNA transcript. The latter effect appeared to derive, at least in part, from suppression of ANP gene transcription. Of interest, both the reduction in mRNA levels and the inhibition of transcriptional activity were amplified by simultaneous treatment with retinoic acid, suggesting that heterodimerization of liganded 1,25(OH)2D3 receptor and retinoic acid receptor (likely retinoid X receptor) may underlie the inhibitory mechanism in the cardiac myocyte. Neither the secretory effect nor the effect on transcription proved to be calcium dependent. 22-Oxacalcitriol, a nonhypercalcemic analogue of 1,25(OH)2D3, was equally effective in suppressing ANP mRNA levels and transcription of the gene. These findings add to a growing body of data that imply an important role for 1,25(OH)2D3 in the regulation of cardiovascular function.