Prostanoids comprising prostaglandins (PGs) and thromboxanes have been shown to play physiological and pathological roles in zebrafish. However, the molecular basis of zebrafish prostanoid receptors has not been characterized to date. Here, we demonstrate that there exist at least six 'relaxant' (Gs-coupled) prostanoid receptors in zebrafish; one PGI(2) receptor IP and five PGE(2) receptors comprising two EP2 (EP2a and EP2b), and three EP4 receptors (EP4a, EP4b and EP4c). In contrast, we failed to find a zebrafish PGD(2) receptor with any structure and/or character similarities to the mammalian DP1 receptor. [H-3]iloprost, a stable IP radioligand, specifically bound to the membrane of cells expressing zebrafish IP with a Kd of 42 nM, and [H-3]PGE(2) specifically bound to the membranes of cells expressing zebrafish EP2a, EP2b, EP4a, EP4b and EP4c with a Kd of 6.9, 6.0, 1.4, 3.3 and 1.2 nM, respectively. Upon agonist stimulation, the 'relaxant' prostanoid receptors showed intracellular cAMP accumulation. The responsiveness of these zebrafish receptors to subtype-specific agonists correlated with their structural conservation to the corresponding receptor in mammals. RT-PCR analysis revealed that the six zebrafish prostanoid receptors show unique tissue distribution patterns; each receptor gene may hence be under unique transcriptional regulation. This work provides further insights into the diverse functions of prostanoids in zebrafish. (C) 2013 Elsevier Inc. All rights reserved.