Expression of the human atrial natriuretic peptide (hANP)gene is controlled by a series of positive and negative cis-acting regulatory elements present in the 5' flanking sequences (5'FS) of the gene. Positive elements located between -1150 and -222, relative to the transcription start site, appear to be responsible for the major portion of ANP gene expression in neonatal rat cardiac atrial cells. While neonatal ventricular cardiocytes, at a qualitative level, seem to employ regulatory signals similar to their atrial counterparts, they do so with reduced efficiency. Expression of the hANP gene in nonmyocardial cells is limited by the presence of silencer elements in the distal (-2593 to -1150) and proximal (-222 to the CAP site) 5'FS. Further characterization of a 64-base pair cardiac-specific element (-410 to -332), described previously, revealed that a core sequence of 40 base pairs is required for functional activity. This core sequence includes a previously defined DNAse-I footprint region flanked by two GC-rich segments arranged in an inverted repeat-like array. These findings suggest that the disparity in atrial vs. ventricular cardiocyte expression of the ANP gene reflects differences that are largely quantitative in nature, while differences in myocardial vs. nonmyocardial cells result from fundamental qualitative differences in the way these cells recognize and use the regulatory elements present within the 5'FS.