The effects of splenectomy and sympathetic blockade on cardiac output (Q̇) response to mild to moderate exercise were studied in chloralose-anesthetized vagotomized dogs during electrically-induced muscular work, which increased oxygen consumption (V̇O<sub>2</sub>) 4- to 5-fold. Splenectomy reduced the exercise factor (ΔQ̇/ΔV̇O<sub>2</sub> ratio) from 5.86 ± 0.43 (SE) to 3.30 ± 0.34 (p < 0.001), while left nephrectomy had no effect. The exercise factor was not affected by β-adrenoceptor blockade produced by propranolol or practolol, but was reduced by mecamylamine. The spleen did not increase cardiac output by displacing blood into the general circulation because pulmonary artery wedge pressure did not change during exercise in normal dogs. Furthermore, cardiac output was increased 35 ± 6% after intraportal infusion of splenic venous blood obtained during exercise, whereas arterial blood infusion increased cardiac output only 10%. We conclude that the cardiac output rise during exercise in part is caused by the inotropic action of a splenic noncatecholamine substance, which is released by sympathetic stimulation.