Cardiac myocytes secrete a family of natriuretic and diuretic peptides, including atrial natriuretic factor (ANF) and brain natriuretic peptide or the rat hormone isoANF. These peptides share structural and functional similarities, but their respective physiological roles have yet to be elucidated. Differential expression of natriuretic peptide genes may reflect distinct physiological or pathophysiological functions for these peptides. To test this hypothesis, we have determined the sites of expression of the ANF and isoANF genes in rat tissues using polymerase chain reaction amplification of ANF and isoANF transcripts. Like ANF mRNA, isoANF mRNA was detected in all heart compartments, and the transcription initiation sites of the isoANF gene, determined from primer extension experiments, were identical in atria and ventricles. In the adult heart, the ventricular isoANF mRNA concentration is only 3 times lower than in atria, and in sharp contrast to ANF, the isoANF gene is constitutively expressed in ventricles during postnatal development. Since ventricles are at least 20 times larger than atria, this implies that isoANF is mostly a ventricular hormone, whereas ANF is essentially an atrial hormone in normal adult hearts. Low levels of isoANF mRNA were found in few extracardiac tissues, including hypothalamus, brain, lung, and aorta. IsoANF transcripts were more abundant than ANF transcripts in aorta, whereas ANF and isoANF mRNA levels were similar in lung. In brain and hypothalamus, ANF transcripts were only 7- and 2-fold greater than isoANF transcripts. The presence of isoANF transcripts in brain and hypothalamus suggests that isoANF is the rat homolog of the human hormone brain natriuretic peptide. Thus, the expression of these two natriuretic peptide genes is not coordinated, suggesting that ANF and isoANF may play different physiological roles in cardiac and extracardiac tissues.