Background. L-arginine, the sole substrate for the Nitric Oxide Synthase (NOS) enzyme in producing NO, is also a substrate for arginase. Methods. We examined normal feline hearts and hearts with compensated left ventricular hypertrophy (LVH) produced by ascending aorta banding. Using Western blot analysis, we examined the abundance of arginase isozymes in crude homogenates and isolated cardiac myocytes obtained from the left ventricles of normal and LVH hearts. We examined the functional significance of myocyte arginase via measurement of shortening and intracellular calcium in isolated myocytes in the presence and absence of borono-ethyl chloride (BEC), a specific pharmacologic inhibitor of arginase. Results. Both arginase I and II were detected in crude myocardial homogenates, but only arginase I was present in isolated cardiac myocytes. Arginase I was downregulated in LVH compared to normal. Inhibition of arginase with BEC reduced fractional shortening, +dL/dt, -dL/dt, and the peak of the [Ca²⁺]_i transient in normal myocytes, but did not affect these parameters in LVH myocytes. These negative inotropic actions of arginase inhibition were associated with increases in cGMP generation. Conclusions. These studies indicate that only arginase I is present in cardiac myocytes where it tends to limit NO and cGMP production with the effect of supporting basal contractility. In experimental LVH induced by pressure overload, our studies demonstrate reduced arginase I expression and reduced functional significance, allowing greater arginine substrate availability for NO/cGMP signaling.