The regulation of rat aorta vascular tone involves various factors, including endothelium-derived hyperpolarization factor (EDHF), nitric oxide (NO), prostaglandins, and sensory nerves. While these elements can function independently, their pathways intersect at various points, complicating the assessment of their individual contributions. The aim of this study was to establish the numerical contributions of EDHF, NO, prostaglandins, and also the effect of the sensory nerve on acetylcholine-induced relaxation on the background of phenylephrine preconstriction using contraction and relaxation measurements in Wistar rat thoracic aorta. EDHF, whose action is mediated through potassium channels, emerges as a crucial regulator. Blockage of inward rectifier potassium (KIR) channels integral to EDHF significantly abolishes 50% of the relaxation amplitude in comparison to control conditions. Endothelial TRPV4 channel, exhibiting a fine-tuning role, contributes to a 25% reduction in the amplitude of acetylcholine-induced relaxation in comparison to control relaxation. NO demonstrates its vasodilatory prowess, with NO blockage eliminating 77% of the residual relaxation effect after KIR blockage. Blockage of prostaglandin functions, modulated by cyclooxygenase 1, reduces relaxation by 44% in comparison to control relaxation. Desensitization of sensory nerves with capsaicin, shows a minor yet significant role, in the reduction of acetylcholine-induced relaxation amplitude by 10%. In conclusion, we established that the main element of acetylcholine-induced relaxation is EDHF with approximately 50% of relaxation amplitude depending on it.