Background - Experimental and clinical evidence suggests that angiotensin II may be an important mediator of cardiac hypertrophy in response to hemodynamic stress. We investigated the effect of genetic variation in angiotensin-converting enzyme (ACE) on the development of cardiac hypertrophy and left ventricular (LV) dysfunction in response to volume overload. Methods and Results - Male heterozygous ACE knockout (1/0) and wild-type (1/1) mice were studied 4 weeks after the creation of an aortocaval fistula (ACF). The LV weight/body weight ratio increased 74% in ACF versus sham-operated control mice but did not differ between genotypes. Echocardiographic circumferential stress versus rate-corrected velocity of circumferential shortening curves demonstrated depressed LV function in ACF versus sham-operated mice but no difference between genotypes. LV ACE activity was higher in 1/1 versus 1/0 mice and in ACF versus sham-operated mice, and it increased significantly more in the 1/1 versus the 1/0 mice after ACF (P<0.001 for effect of genotype, ACF/sham operation, and interaction term). LV angiotensin II was higher in ACF versus sham-operated mice but did not differ between genotypes, despite 3-fold higher LV ACE activity in ACF 1/1 versus ACF 1/0 mice. Conclusions - ACE underexpression does not prevent cardiac hypertrophy or LV dysfunction in response to volume overload. LV angiotensin II is unaffected by ACE genotype, both at baseline and after volume overload, indicating that the heart can maintain angiotensin II levels across a broad range of genetic ACE variation under both physiological and pathophysiological conditions.