We investigated the effects of epoxyeicosatrienoic acids (EETs) on ion transport in the polarized renal distal tubular cell line, MDCK C7. Of the four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) studied, only apical, but not basolateral, application of 5,6-EET increased short circuit current (Isc) with kinetics similar to those of arachidonic acid. The ion transport was blocked by preincubation with the cyclooxygenase inhibitor indomethacin or with the chloride channel blocker NPPB. Further, both a Cl--free bath solution and the Ca²⁺ antagonist verapamil blocked 5,6-EET-induced ion transport. Although the presence of the PGE₂ receptors EP2, EP3, and EP4 was demonstrated, apically added PGE₂ was ineffective and basolaterally added PGE₂ caused a different kinetics in ion transport compared to 5,6-EET. Moreover, PGE₂ sythesis in MDCK C7 cells was unaffected by 5,6-EET treatment. GC/MS/MS analysis of cell supernatants revealed the presence of the biologically inactive 5,6-dihydroxy-PGE₁ in 5,6-EET-treated cells, but not in control cells. Indomethacin suppressed the formation of 5,6-dihydroxy-PGE₁. 5,6-epoxy-PGE1 the precursor of 5,6-dihydroxy-PGE₁, caused a similar ion transport as 5,6-EET. Cytochrome P450 enzymes homolog to human CYP2C8, CYP2C9, and CYP2J2 protein were detected immunologically in the MDCK C7 cells. Our findings suggest that 5,6-EET affects Cl- transport in renal distal tubular cells independent of PGE₂ but by a mechanism, dependent on its conversion to 5,6-epoxy-PGE₁ by cyclooxygenase. We suggest a role for this P450 epoxygenase product in the regulation of electrolyte transport, especially as a saluretic compound acting from the luminal side of tubular cells in the mammalian kidney.