In a genetically engineered mouse line with disruption of type 5 adenylyl cyclase (AC5^−/−), a major cardiac isoform, there was no compensatory increase in other isoforms of AC in the heart. Both basal and isoproterenol (ISO)-stimulated AC activities were decreased by 30% to 40% in cardiac membranes. The reduced AC activity did not affect cardiac function (left ventricular ejection fraction [LVEF]) at baseline. However, increases in LVEF after ISO were significantly attenuated in AC5^−/− (P<0.05, n=11). Paradoxically, conscious AC5^−/− mice had a higher heart rate compared with wild-type (WT) mice (613±8 versus 523±11 bpm, P<0.01, n=14 to 15). Muscarinic agonists decreased AC activity, LVEF, and heart rate more in WT than in AC5^−/−. In addition, baroreflex-mediated, ie, neuronally regulated, bradycardia after phenylephrine was also attenuated in AC5^−/−. The carbachol-activated outward potassium current (at −40 mV) normalized to cell capacitance in AC5^−/− (2.6±0.4 pA/pF, n=16) was similar to WT (2.9±0.3 pA/pF, n=27), but calcium (Ca²⁺)-mediated inhibition of AC activity and Ca²⁺ channel function were diminished in AC5^−/−. Thus, AC5^−/− attenuates sympathetic responsiveness and also impairs parasympathetic and Ca²⁺-mediated regulation of the heart, indicating that those actions are not only regulated at the level of the receptor and G-protein but also at the level of type 5 AC.