Atrial natriuretic peptide (ANP) exhibits relaxant and growth-inhibiting effects on vascular smooth muscle cells (VSMCs). To obtain ANP gene expression in VSMCs, we built a recombinant adenovirus containing the ANP cDNA controlled by the adenovirus major late promotor (AdMLP-ANP). After pulmonary VSMC treatment with AdMLP-ANP at a multiplicity of infection ranging from 5 to 100 TCID50/cell, immunoreactive ANP was detectable in the cell culture medium at a level that reached 101 ± 27 pmol/well after 2 days. The newly expressed ANP was biologically active, as evidenced by its ability to induce cyclic guanosine monophosphate accumulation in target cells and to mimic the effect of exogenous ANP (10-8 to 10-7 mol/L). Cell growth and survival of AdMLP-ANP-infected cells were decreased and were associated with the promotion of VSMC apoptosis. These effects, which occurred at a multiplicity of infection of 10 to 100 TCID50/cell, were observed neither in cells infected with the control adenoviral constructs (AdMLP-ßGAL and AdMLP-gD) nor in cells treated with exogenous ANP (10-7 to 10-6 mol/L). These results showing VSMC apoptosis in response to ANP gene expression may have important implications for the prevention of vascular remodeling by gene therapy.