The current study examined the contributions of angiotensin-converting enzyme (ACE) vs. chymase to angiotensin II (ANG II) generation in membrane preparations from left ventricles of humans, dogs, rabbits, and rats and from total heart of mice. ACE and chymase activity were measured in membrane preparations extracted with low or high detergent (LD and HD, respectively) concentrations. We hypothesized that ACE, which is membrane bound in vivo, would be preferentially localized to the HD preparation, whereas chymase, which is localized to the cytoplasm and cardiac interstitium, would be localized to the LD preparation. In human heart, ACE activity was 16-fold higher in the HD than in the LD preparation, whereas chymase activity was 15-fold higher in the LD than in the HD preparation. Total ANG II formation was greater in human heart [15.8 ± 3.4 (SE) μmol ANG II ⋅ g⁻¹ ⋅ min⁻¹] than in dog, rat, rabbit, and mouse hearts (3.90 ± 0.35, 0.41 ± 0.02, 0.61 ± 0.07, and 1.16 ± 0.08 μmol ANG II ⋅ g⁻¹ ⋅ min⁻¹, respectively, P < 0.05, by analysis of variance). ANG II formation from ACE was higher in mouse heart (1.09 ± 0.05 μmol ANG II ⋅ g⁻¹ ⋅ min⁻¹,P < 0.001) than in rabbit, human, dog, and rat hearts (0.55 ± 0.06, 0.34 ± 0.01, 0.32 ± 0.06, and 0.31 ± 0.02 μmol ANG II ⋅ g⁻¹ ⋅ min⁻¹, respectively). In contrast, chymase activity was higher in human heart (15.3 ± 3.4 μmol ANG II ⋅ g⁻¹ ⋅ min⁻¹) than in dog, rat, rabbit, and mouse hearts (3.59 ± 0.29, 0.10 ± 0.01, 0.06 ± 0.01, and 0.07 ± 0.01 μmol ANG II ⋅ g⁻¹ ⋅ min⁻¹, respectively). Our results demonstrate important species differences in the pathways of intracardiac ANG II generation. Chymase predominated over ACE activity in human heart, accounting for extremely high total ANG II formation in human heart compared with dog, rat, rabbit, and mouse hearts.