The activator protein 1 (AP-1) transcriptional complex, containing Jun and Fos proteins, is involved in regulating many cellular processes such as proliferation and differentiation. However, little is known about a direct relationship between AP-1 activities and cardiomyocyte hypertrophy. To elucidate the roles of myocardial AP-1 activities, dominant negative mutant of c-Jun (DNJun) was overexpressed in cultured rat neonatal ventricular myocytes by adenovirus vector to abrogate endogenous AP-1 activation. Cardiomyocytes were treated with 100 nmol/L endothelin 1 (ET) and 10 &mgr;mol/L phenylephrine (PE) to induce myocardial cell hypertrophy. Both ET and PE significantly enhanced AP-1 DNA binding activities (3.4-fold by ET and 4.8-fold by PE at 3 hours,P<0.01). At 48 hours after stimulation, ET and PE significantly increased incorporation of3H-phenylalanine (1.4-fold by ET and 1.5-fold by PE,P<0.01), cell size (2.3-fold and 2.5-fold,P<0.01), and mRNA expression of atrial natriuretic peptide (ANP; 1.9-fold and 1.8-fold,P<0.01) and brain natriuretic peptide (BNP; 1.6-fold and 1.6-fold,P<0.01). Adenovirus carrying DNJun prevented the transcriptional activation of the AP-1 by ET and PE, using AP-1 reporter enzyme firefly luciferase assay. Moreover, DNJun prevented the increase in incorporation of3H-phenylalanine, cell size, and the mRNA expression of ANP and BNP by ET and PE. In conclusion, we provide the first evidence that DNJun inhibits cardiomyocyte hypertrophy through inhibition of AP-1 transcriptional activity.