The signaling cascades that activate transcription factors during cardiac hypertrophy are largely unknown. To evaluate the mechanisms for GATAff4 and activator protein-1 (AP-1) activation, isolated perfused rat hearts were exposed to elevated wall stretch by inflating a left ventricular balloon. Gel mobility shift assays were used to analyze the transacting factors that interact with the GATA or the AP-1 motifs of the B-type natriuretic peptide (BNP) promoter. Direct wall stretch for 30 min produced a twofold increase (P<0.001) in left ventricular BNP GATAff4- but not GATAff5- and GATAff6-binding activity. In addition, increased BNP AP-1-binding activity and the presence of c-fos were demonstrated in wall-stretch-stimulated ventricles compared with unloaded ventricles. The mixed endothelin-1 ET_A/ET_B receptor antagonist bosentan and the angiotensin II (Ang II) type 1 (AT₁) receptor antagonist CV-11974 completely inhibited the wall-stretch-induced increase in left ventricular BNP GATAff4 and AP-1 activity. Infusions of ET-1 and Ang II in the absence of wall stretch also stimulated BNP GATAff4-binding activity (P<0.01). These results show that ET-1 and Ang II are required for the stimulation of GATAff4 and AP-1 DNA-binding activity in response to direct left ventricular wall stretch.