Using a primary culture of pneumocytes derived from neonatal rat lungs, we investigated the synthesis and secretion at transcriptional and peptide levels of pulmonary rat(r) atrial natriuretic peptide (ANP). Total RNA extracted from pneumocytes contained a hybridizing RNA band of the same size as atrial rANP mRNA. Immunoreactive (IR)-rANP content in pneumocytes was 0.5% of that in atrial myocytes, and 8.6% of that in ventricular myocytes, while the secretory rate from pneumocytes was about 7% of atrial and ventricular myocytes. Triiodothyronine (T 3, 5 × 10 −10 to 5 × 10 −8 M), dexamethasone and testosterone (5 × 10 −9 to 5 × 10 −8 M) significantly stimulated the synthesis of IR-rANP by pneumocytes in a dose-dependent manner. However, the stimulatory effect exerted by T 3 on rANP synthesis, unlike in the case of cardiocytes, was much more potent than that of dexamethasone, as evidenced by the significant difference in potency at both transcriptional and peptide levels. The present study suggests that ANP secreted from lungs may at least in part contribute to circulating ANP pool, and that the tissue-dependent difference of sensitivity to thyroid hormone may play an important role in the regulation of developmental ANP gene expression in mammalian lungs.