Abstract Vasodilation, an important response in neurogenic inflammation, involves release of Substance P (SP) from the sensory nerve endings. It is now well known that SP causes edema formation and vascular relaxation in nondental tissues, however, the SP vasodilatory mechanism in the dental pulp is not completely understood. Endothelium-dependent relaxation is mediated by nitric oxide (NO) release with consecutive intracellular cyclic-GMP elevation in many vascular preparations. Recently, it has been shown in different vascular systems that SP-induced vasodilation is mediated by cyclic-GMP production through different pathways involving endothelial NO or direct endothelial-independent pathways. In the present study, the role of endothelial NO in SP induced vasodilation in the dental pulp was investigated to better understand the inflammatory mechanisms. Freshly extracted bovine dental pulp was used to measure NO production. Sodium nitroprusside (SNP), L-NAME and SP were utilized to induce and to inhibit NO production in endothelial cells. Released NO byproducts were measured with chemiluminescence assay technique. The present data demonstrate that SP induces NO production by activating NOsynthase (NOS) in endothelial cells. The NOS inhibitor L-NAME blocks NO production completely. In conclusion, in the bovine dental pulp, SP-induced vascular relaxation can be mediated by inducing NOS, and subsequently NO production in endothelial cells.