A cDNA copy of the message encoding rat atrial natriuretic factor (ANF) has been cloned in Escherichia coli, and its nucleotide sequence was determined. ANF appears to be synthesized as a larger precursor, atrial pronatriodilatin. The cDNA has an open reading frame potentially encoding a protein of 152 amino acids, of which the first 24 amino acids strongly resemble a signal sequence. This is followed by a sequence with 80% homology to a second vasoactive protein, porcine cardiodilatin. The ANF peptide is contained in the COOH-terminal portion of the protein. The DNA sequence corresponding to human ANF is also presented and displays a high degree of homology to its rat counterpart. These data provide further evidence for the expression in cardiac atria of a multifactor system that may contribute to the regulation of blood pressure and extracellular fluid volume.