A complementary DNA (cDNA) encoding eel atrial natriuretic peptide (ANP) precursor was specifically amplified from eel atrial mRNAs by rapid-amplification polymerase chain reaction. The sequence analysis of the cDNA using multiple clones revealed that the preproANP consists of 140 amino acid residues carrying a signal sequence at its N-terminus and a mature ANP at its C-terminus. An additional glycine residue was attached to the C-terminus of previously isolated eel ANP. The glycine residue may be used for amidation of the C-terminus or removed after processing. The cleavage site of a signal peptide with 22 amino acid residues was confirmed by isolation of proANP protein from eel atria. The proANP sequence deduced from the cDNA was also confirmed for 71% of the isolated protein. Sequence comparison with other natriuretic peptides revealed that eel ANP is more similar to mammalian ANP than to B-type natriuretic peptide (BNP) at both amino acid and nucleotide sequence levels. The eel ANP gene was a single copy gene as shown by Southern blot analysis. Northern blot analysis showed that eel ANP mRNA is approximately 0.8 kb in size and exclusively detected in the atrium. Thus, eel ANP is a true atrial hormone judging from both the sequence and the site of production. However, reverse transcription-polymerase chain reaction detected ANP message in the brain, gill, cardiac ventricle, red body of swim bladder (rete mirabilis), intestine, head kidney (including interrenal and chromaffin tissues) and kidney. Most of these tissues are involved in ion and/or gas exchange in fishes.