Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which act as cardiac hormones, are produced mainly by the atrium and ventricle, respectively, and are involved in body fluid homeostasis and blood pressure control. The ANP and BNP gene expressions are markedly augmented in ventricles of patients with a wide variety of cardiovascular diseases. It has been demonstrated that the ANP and BNP genes are tightly linked on mouse chromosome 4 and on the distal short arm of human chromosome 1. However, the precise physical map of the ANP and BNP genes has never been elucidated. In the present study, we characterized the genomic DNA fragment containing the ANP and BNP genes in mice and humans. Three genomic DNA clones harboring the entire mouse BNP gene were isolated from a 129/Sv mouse genomic DNA library. Nucleotide sequence analysis revealed that a phage clone (lambda mBNP #3) contains at its 3'-end the 5'-flanking region and the first 209-bp sequence of the first exon of the mouse ANP gene. In mice, the BNP gene was located about 12 kb upstream of the ANP gene. By polymerase chain reaction, we isolated an approximately 11-kb human genomic DNA fragment containing the third exon of the BNP gene and the first and second exons of the ANP gene. In humans, the BNP gene was located upstream of the ANP gene, approximately 8 kb apart. The present study provides the direct evidence that the ANP and BNP genes are organized in tandem in the mouse and human genomes.