The identification of estrogen-responsive genes in the heart, is necessary to understand estrogen-induced changes in cardiac function. Using Delta RNA fingerprinting, we demonstrate that a single injection of estradiol benzoate (50 microg, s.c.) revealed mRNA species that were elevated, down-regulated, or were unaffected in the heart tissue of ovariectomized female rats. One of the upregulated genes was identified, by cloning and sequencing, to have 95.8% (230/240) identity with the 3' end of the rat ant1 gene encoding the mitochondrial adenine nucleotide translocator, ANT1. Using the isolated ANT1 cDNA (280 bp) as a probe in Northern analysis, estrogen was shown to upregulate the expression of cardiac ANT1, by at least 3-fold in female rats, from as early as 1 h to as long as 24 h. In contrast, estrogen treatment had no effect on ANT1 expression in heart tissue from male rats. RNA yields were low in rat atria and no transcript was detectable by Northern analysis. Using primers specific to the known rat ANT1 gene, the estrogen upregulation of the cardiac ANT1 transcript in female rat was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR); a predicted product of 249 bp was obtained and this was stimulated by at least 3-fold upon estrogen treatment for 24 h.