Induction of cyclooxygenase-2 (COX-2) in the renal pelvic wall increases prostaglandin E₂ (PGE₂) leading to stimulation of cAMP production which results in substance P (SP) release and activation of renal mechanosensory nerves. The subtype of PGE receptors involved, EP2 and/or EP4, was studied by immunohistochemistry and renal pelvic administration of agonists and antagonists of EP2 and EP4 receptors. EP4 receptor-like immunoreactivity (LI) was colocalized with calcitonin gene-related peptide (CGRP)-LI in dorsal root ganglia (DRGs) at Th₉-L₁ and in nerve terminals in the renal pelvic wall. Th₉-L₁ DRG neurons also contained EP3 receptor-LI and COX-2-LI, each of which was colocalized with CGRP-LI in some neurons. No renal pelvic nerves contained EP3 receptor-LI and only very few nerves COX-2-LI. The EP1/EP2 receptor antagonist AH6809, 20 µM, had no effect on SP release produced by PGE₂, 0.14 µM, from an isolated rat renal pelvic wall preparation. However, the EP4 receptor antagonist L-161,982, 10 µM, blocked the SP release produced by the EP2/EP4 receptor agonist butaprost, 10 µM, 12 ± 2 vs. 2 ± 1 and PGE₂, 9 ± 1 vs. 1 ± 0 pg/min. The SP release by butaprost and PGE₂ was similarly blocked by the EP4 receptor antagonist AH23848, 30 µM. In anesthetized rats, the afferent renal nerve activity (ARNA) responses to butaprost, 700 ± 100, and PGE₂, 780 ± 100 %^.sec (area under the curve of ARNA vs. time) were unaffected by renal pelvic perfusion with AH6809. However, 1 µM L-161,982 and 10 µM AH23848 blocked the ARNA responses to butaprost by 94 ± 5 and 78 ± 10%, respectively, and to PGE₂ by 74 ± 16 and 74 ± 11%, respectively. L-161,982 also blocked the ARNA response to increasing renal pelvic pressure 10 mmHg, 85 ± 5%. Conclusion: PGE₂ increases renal pelvic release of substance P and ARNA by activating EP4 receptors on renal sensory nerve fibers.