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Purpose: The retina contains a number of vasoactive neuropeptides and corresponding receptors, but the role of these neuropeptides for tone regulation of retinal arterioles has not been studied in detail. Methods: Porcine arterioles with preserved perivascular retinal tissue were mounted in a wire myograph, and the tone was measured after the addition of increasing concentrations of bradykinin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), substance P (SP), calcitonin gene‐related peptide (CGRP) and brain natriuretic peptide (BNP). The experiments were performed during inhibition of the synthesis of nitric oxide (NO), prostaglandins and dopamine and were repeated after removal of the perivascular retinal tissue. Results: Bradykinin, VIP and CGRP induced significant concentration‐dependent dilatation and NPY significant concentration‐dependent contraction of the arterioles in the presence of perivascular retinal tissue (p < 0.03 for all comparisons) but not on isolated arterioles. BNP and SP had no effect on vascular tone. The NOS inhibitor L‐NAME reduced bradykinin‐ and VIP‐induced relaxation (p < 0.001 for both comparisons), whereas none of the other inhibitors influenced the vasoactive effects of the studied neuropeptides. Conclusion: The effects of neuropeptides on the tone of retinal arterioles depend on the perivascular retinal tissue and may involve effects other than those mediated by nitric oxide, prostaglandins and adrenergic compounds. Investigation of the mechanisms underlying the vasoactive effect of neuropeptides may be important for understanding and treating retinal diseases where disturbances in retinal flow regulation are involved in the disease pathogenesis. [ABSTRACT FROM AUTHOR]