Primary cultures of atrial myocytes were prepared from newborn rats and maintained for 8 days in complete serum-free medium. The culture content of immunoactive atrial natriuretic peptide (ANP) increased from 10 to 25 ng/culture during this time. The cells released immunoactive ANP at a rate of 2 to 3% of culture content per hour in a linear fashion for at least 6 hours. When analyzed by gel filtration the major immunoactive material released by and contained within the cells displayed a molecular weight of approximately 15,000 daltons. The medium and cellular ANP-related peptides were further shown to be indistinguishable by reversed-phase HPLC. When the 15,000 dalton material was incubated with rat serum it was converted to ANP-related material possessing a molecular weight of approximately 3,000 daltons. These results suggest that under basal conditions, atrial myocytes release a large molecular weight form of ANP that is converted in the circulation to a low molecular weight form of ANP, which has been previously identified in plasma.