Atrial natriuretic peptide (ANP) is a cardiac hormone that elicits a profound diuresis, natriuresis, and hypotension. As a preliminary study toward ANP gene therapy of cardiovascular disorders, we have cloned a cDNA for mouse preproANP and carried out expression studies in muscle cells, The expression cassette, which was flanked by ITRs from AAV-8, consisted of HCMV IE enhancer/promoter, preproANP gene, and polyadenylation signal from bovine growth hormone. We transfected this expression vector into primary skeletal myoblasts and examined the following points: (1) secretion of immunoreactive ANP, (2) biological activity, and (3) nature of secreted ANP(s). The conditioned media from cells transfected with ANP vector had significantly higher levels of irANP in comparison to mock control. The secreted irANP had biological activity as confirmed by the elevated level of intracellular cGMP in human umbilical vein endothelial cells. Reverse-phase HPLC analysis showed that the processed form of ANP was the predominant form. These results demonstrate that preproANP gene could be ectopically expressed and correctly processed in skeletal myoblasts, which has implications for development of muscle-based ANP gene therapy.