Expression of the human atrial natriuretic peptide (hANP) gene was examined in tissues of 19 to 28-week-old human fetuses. As expected the fetal atria express the hANP gene at a high level, accruing substantial quantities of ANP immunoreactivity and ANP mRNA. The neonatal ventricle also expresses the hANP gene at a significant level. ANP mRNA levels in the ventricle were, on the average, about 20% of those in the right atria. ANP immunoreactivity in ventricle was less that 5% of that in the right atria, suggesting important differences in the way these tissues synthesize and store the ANP peptide. Much lower levels of hANP transcripts were also detected in the lung and aortic arch. Analysis of the 5' termini of cardiac hANP transcripts using three independent techniques suggests the presence of two transcription start sites. A major start site is located approximately 28 basepairs downstream from the primary TATA sequence. A second minor start site is positioned about 110 basepairs further upstream. Immunocytochemistry and in situ hybridization analysis indicate that the hANP gene is expressed in a homogeneous distribution throughout the atrial myocardium. Diffuse low level expression is also present within the ventricular myocardium. In addition, there are scattered foci of increased expression in the ventricle which tend to be concentrated in the subendocardium of that organ. These findings indicate that the developing human ventricles as well as the atria possess the capacity to express the hANP gene at a substantial level and suggest that the ventricle may contribute significantly to the circulating pool of plasma ANP.