The presence of immunoreactive atrial natriuretic polypeptide (ANP) has been demonstrated in fetal atria early in gestation but further definition of fetal ANP has not been reported. To characterize the principal molecular forms of fetal ANP and to compare fetal ANP to that of the adult of the same species, we extracted the atria of pregnant adult and 20-day fetal rats, the hearts of 14-day fetuses, and intact 12-day fetuses in 1 M acetic acid. Tissue collected from littermates was pooled. We measured ANP by radioimmunoassay before and after gel filtration on Sephadex G-75 in each group. ANP concentrations (means +/- 1 SD) in ng/mg protein and ng/animal were 1296 +/- 505 and 7707 +/- 1877 in adult atria (n = 17), 174 +/- 44 and 62 +/- 13 in 20-day fetal atria (n = 7), and 33 +/- 5.3 and 3.7 +/- 0.9 in 14-day fetal hearts (n = 6), respectively. Acid extracts from intact 12-day fetuses did not dilute in parallel to the standard curve; therefore, concentrations of ANP for the 12-day fetuses are not reported. ANP concentration rose from the 20-day fetus to the adult (p less than 0.0001). The major species of ANP eluting from the Sephadex column had an apparent molecular weight of 16 K in all groups. We conclude: 1) ANP is present in the fetus shortly after the completion of organogenesis; 2) 16 K ANP is the principal intracardiac species in the fetus and the adult; and 3) the existence of ANP soon after cardiac development suggests a possible role for ANP in fetal blood pressure and sodium and water homeostasis.