The roles of different mediators in the sympathetic regulation of the pig and dog spleens were investigated using a preparation with intact vascular perfusionin vivo. Sympathetic nerve stimulation caused overflow of neuropeptide Y‐like immunoreactivity (NPY‐LI) and noradrenaline (NA), arterial vasoconstriction, increase in venous blood flow and haematocrit. The dog spleen responded to single impulse stimulation, whereas more prolonged stimulation was required to elicit vascular responses in the pig spleen. Furthermore, the maximal splenic capacitance response was about 10 times larger in the dog than in the pig. After depletion of neuronal NA content by reserpine combined with preganglionic denervation, about 70% of the splenic arterial vasoconstrictor responses in the dog and pig still remained at 5 Hz stimulation. Fifty per cent of the capacitance response evoked by nerve stimulation still remained in the pig while in the dog spleen the capacitance response was virtually abolished after reserpine. The stimulation‐evoked overflow of NPY‐LI in pig spleen was increased several fold after reserpine treatment as compared to controls reaching levels in the venous effluent where exogenous NPY evokes vasoconstriction. In the dog spleen, overflow of NPY‐LI was only observed after reserpine. Administration of NA caused arterial vasoconstriction with an initial increase in venous blood flow while NPY mainly reduced arterial blood flow.It is concluded that NA is involved in both the splenic arterial vasoconstriction and the capacitance responses while a non‐adrenergic splenic vasoconstriction at least in the pig may be medi