Despite intensive investigation, the role of nitric oxide (NO) in cholinergic modulation of myocardial contractility remains unresolved. The left anterior descending coronary artery of 34 anesthetized, open-chest dogs was perfused via an extracorporeal circuit. Segmental shortening (SS) was measured with ultrasonic crystals and coronary blood flow (CBF) was measured with an ultrasonic flow transducer. An intracoronary infusion of ACh (20 µg/min) was performed, with CBF held constant, under baseline and during dobutamine, CaCl₂, or amrinone at doses increasing SS by ~50% (10 µg/min, 15 mg/min, and 300 µg/min ic, respectively). ACh-induced responses during dobutamine were also assessed following treatment with the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 300 µg/min ic for 15 min). The effects of sodium nitroprusside (SNP; 80 µg/min ic), an exogenous NO donor, bradykinin (2.5 µg/min ic), a nonmuscarinic releaser of endothelial NO, and bilateral vagal stimulation (before and after L-NAME) were evaluated during dobutamine. ACh had no effect on SS under baseline or during CaCl₂, but it decreased SS during dobutamine or amrinone (23 ± 4% and 30 ± 5%, respectively). Vagal stimulation also reduced SS during dobutamine. L-NAME did not alter the ACh- or vagal-induced decreases in SS during dobutamine. Neither SNP nor bradykinin affected SS during dobutamine. In conclusion, ACh and vagal stimulation have a negative inotropic effect during stimulation of the -adrenergic receptors that is independent of NO. The persistence of this effect during amrinone suggests that a mechanism downstream from adenylate cyclase is involved.