Previous work from this laboratory has shown that amiodarone induces alveolar epithelial cell apoptosis that was abrogated by antagonists of angiotensin II. In this study, amiodarone up-regulated angiotensinogen mRNA and protein in primary cultures of rat type II pneumocytes and in the human A549 cell line. The mechanism of amiodarone-induced angiotensinogen expression was studied in A549 cells with a human angiotensinogen promoter-luciferase reporter (angiotensinogen/luciferase). Amiodarone (3 microg/ml) induced both angiotensinogen/luciferase and endogenous angiotensinogen mRNA; the latter was completely blocked by actinomycin-D. Amiodarone did not affect the half-life of endogenous angiotensinogen mRNA. Deletion analyses of angiotensinogen/luciferase identified at least two amiodarone-responsive domains in the angiotensinogen promoter between -350 to -260 bp and -203 to -46 bp. DNA/Protein array and electrophoretic mobility shift assays showed that amiodarone increases DNA binding of both activation protein-1 and STAT-5 transcription factors. Site-directed mutagenesis of three IL-6-responsive signal transducer activator of transcription (STAT) binding sites within the amiodarone-response domains had no effect on amiodarone-induced angiotensinogen/luciferase expression. In contrast, amiodarone-induced angiotensinogen/luciferase expression was abrogated by a dominant-negative fos construct and was stimulated over five times by c-fos and c-jun expressed together but not separately. Mutagenesis of the activation protein-1 binding site at -15 to -12 bp completely eliminated the response to amiodarone. These data show that activation protein-1 family transcription factors mediate amiodarone-induced angiotensinogen expression in human alveolar epithelial cells and identify an activation protein-1 site, located between the TATA (DNA sequence) box and the transcription initiation site, that is required for the response.