Recent evidence suggests that paracrine signaling agents, such as endothelin (ET), nitric oxide (NO), superoxide (O₂^-), and prostanoids can modulate mammalian renal function by affecting both hemodynamic and epithelial, ionic transport pathways. Since these signaling pathways have been described in fish blood vessels, we hypothesized that they may control salt transport across the gill epithelium--the primary site of ion excretion in marine teleost fishes. We found that ET, the NO-donors sodium nitroprusside and spermine NONOate, and the prostanoid PGE₂ each can produce a concentration-dependent reduction in the short circuit current (Isc) across the isolated opercular epithelium of the killifish (Fundulus heteroclitus), the generally accepted model for the marine teleost gill epithelium. Sarafotoxin S6c was equipotent to ET-1, suggesting that ET_B receptors are involved. Incubation with L-NAME or indomethacin reduced the effect of subsequent addition of SRXS6c by 17% and 89%, respectively, suggesting the presence of an ET to NO and PGE axis. The effects of L-NAME and indomethacin were not additive, but the SOD-mimetic, TEMPOL, reduced the effect of SRXS6c by 34% and pre-incubation with L-NAME, indomethacin, and TEMPOL reduced the SRXS6c response to zero. This suggests a direct role for O₂^- in this axis. COX-2 appears to be the major enzyme involved in this axis because the specific COX-2 inhibitor NS-398 was twice as effective as the COX-1 inhibitor SC560 in inhibiting the SRXS6c effect. The Isc was stimulated by the EP₂ agonist butaprost and inhibited by the EP_1,3 agonist sulprostone, suggesting both stimulatory and inhibitory PGE receptors in this tissue. Carbaprostacyclin (PGI₂ analogue), thromboxane A₂, PGF₂, and PGD₂ did not affect the Isc. Our data are the first to suggest the importance of an ET-stimulated, and NO, O₂^-. and PGE₂ mediated signaling axis that can modify active extrusion of NaCl across the killifish opercular epithelium and, by inference, the marine teleost gill epithelium.