Molecular forms of atrial natriuretic peptide (ANP) were determined by chromatography on Sephadex G50 fine coupled with a radioimmunoassay for rANP(1-28) in left and right atrial extracts and acidified plasmas from 17-, 19- and 21-day-old fetuses and adult female rats. Chromatographic analyses revealed two immunoreactive peaks of rANP with an apparent molecular weight of about 25 and 12 kD, respectively, in all atrial extracts from fetal and adult rats. Only one immunoreactive peak of rANP with an apparent molecular weight of about 3 kD was detected in all fetal plasmas. The 25- and 12-kD ANP molecular forms isolated from fetal atrial tissue and incubated with thrombin for 30 min at 37 degrees C were converted to ANP material with a molecular weight of about 3 kD. These results suggest that the two high molecular weight forms stored in fetal atrial tissue should be precursors of circulating rANP molecules, and the rat fetus should possess the same posttranslational processing of the precursor molecule as the adult rat as early as day 17 of gestation.