Recent studies implicate increased cGMP synthesis as a postreceptor contributor to reduced cardiac sympathetic responsiveness. Here we provide the first evidence that modulation of this interaction by cGMP‐specific phosphodiesterase PDE5A is also diminished in failing hearts, providing a novel mechanism for blunted β‐adrenergic signaling in this disorder. In normal conscious dogs chronically instrumented for left ventricular pressure‐dimension analysis, PDE5A inhibition by EMD82639 had modest basal effects but markedly blunted dobutamine‐enhanced systolic and diastolic function. In failing hearts (tachypacing model), however, EMD82639 had negligible effects on either basal or dobutamine‐stimulated function. Whole myocardium from failing hearts had 50% lower PDE5A protein expression and 30% less total and EMD92639‐inhibit‐able cGMP‐PDE activity. Although corresponding myo‐cyte protein and enzyme activity was similar among groups, the proportion of EMD82639‐inhibitable activity was significantly lower in failure cells. Immunohis‐tochemistry confirmed PDE5A expression in both the vasculature and myocytes of normal and failing hearts, but there was loss of z‐band localization in failing myocytes that suggested altered intracellular localization. Thus, PDE5A regulation of cGMP in the heart can potently modulate β‐adrenergic stimulation, and alterations in enzyme localization and reduced synthesis may blunt this pathway in cardiac failure, contributing to dampening of the β‐adrenergic response.—Senzaki, H., Smith, C. J., Juang, G. J., Isoda, T., Mayer, S. P., Ohler, A., Paolocci, N., Tomaselli, G. F., Hare, J. M., Kass, K. A. Cardiac phosphodiesterase 5 (cGMP‐specific) modulates β‐adrenergic signaling in vivo and is down‐regulated in heart failure. FASEB J. 15, 1718—1726 (2001)