Abstract Prostaglandins E (PGE) and F (PGF) mediate diverse physiological functions via their cell surface receptors – prostaglandin E receptor (EP) subtypes 1, 2, 3 and 4 (EP1; EP2; EP3; EP4) and F receptor (FP). In teleost fishes, PGE was implicated in gill epithelium ion transport, while both PGE and PGF were involved in oocyte maturation, follicular rupture and coordination of reproductive behaviors. However, little is known about the mechanisms behind their actions. In present study, we first identified the full-length ORF cDNA clones of three zebrafish prostaglandin E receptor subtype 1 (zEP1) isoforms – zEP1a, zEP1b and zEP1c – and FP (zFP) from adult ovary. RT-PCR showed that zEP1a, zEP1b and zFP are widely expressed in adult tissues, while zEP1c mRNA expression is mainly confined in brain and kidney. Using a pGL3-NFAT-RE luciferase reporter system, both zEP1a and zEP1b expressed in DF-1 cells were shown to be activated by PGE2 potently while zEP1c and zFP were activated by PGF2a effectively, suggesting that the four receptors are functionally coupled to intracellular Ca2+-signaling pathway. Furthermore, EP1a and EP1b, but not EP1c were suggested to couple to cAMP-PKA signaling pathway using a pGL3-CRE luciferase reporter assay. Although zEP1c might originate as a paralog to zEP1a and zEP1b, its functional coupling to PGF2α instead of PGE2 suggested that zEP1 isoforms might have sub-functionalized in their ligand binding and G protein coupling specificity, in addition to differential tissue distribution. Characterization of these receptors undoubtedly furthered our understanding on the diverse yet highly target-specific responses of prostaglandins in teleosts.