It is well recognized that medullary thick ascending limb (mTAL) cells express the EP-prostanoid, EP₃ receptor which attenuates AVP-dependent NaCl transport. Other studies suggest that renal epithelial cells express an IP-prostanoid, IP receptor coupled to Gs-protein which activates adenylate cyclase, and recently a novel IP receptor which inhibits AVP-stimulated water reabsorption has been reported in rabbit cortical collecting duct (rCCD). The purpose of the present study is to biochemically identify and characterize EP and IP receptor subtypes linked to modulation of cAMP production in freshly isolated suspensions of rat mTAL. The effects of PGE₂, and its agonists, sulprostone (SLP) and butaprost (BTP); and PGI₂ agonists, Iloprost (ILP), Carba-prostacyclin (c-PGI₂) and Cicaprost (CCP) on cAMP production in the absence and presence of AVP were assessed. Pertussis toxin (PT) and bisindolymaleimide (BIS) were used to evaluate the roles of Gi-protein, and protein kinase C (PKC) respectively. The results provide biochemical evidence for the expression of EP₂, or EP₃, EP₄, and novel “IP₃” receptor subtypes in freshly isolated rat mTAL.