We describe the biochemical properties of an eicosanoid-modulated Cl^- channel, and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its open probability (Po). Following a purification protocol involving wheat-germ agglutinin (WGA) -affinity and anion exchange chromatography, the proteins were sequentially inserted into liposomes which were then fused into planar lipid bilayers. Functional and biochemical characterization tests confirm that the Cl^- channel is a 55-kDa glycosylated monomer with voltage- and [Ca²⁺]- independent activity. 5,6- and 8,9-EET decreased the conductance of the native channel (control conductance: 70 ± 5 pS in asymmetrical 50 mM trans / 250 mM cis CsCl) in a concentration-dependent manner, with respective IC₅₀ values of 0.31 and 0.42 µM. These regioisomers similarly decreased the conductance of the purified channel (control conductance value: 75 ± 5 pS in asymmetrical 50 mM trans / 250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the Po of the native channel with respective IC₅₀ values of 0.27 and 0.30 µM, but failed to alter the Po of the purified protein. Thus, we suggest that the effects of these EETs on channel conductance likely result from direct interactions of EET^- anions with the channel pore, whereas the alteration of Po requires a lipid environment of specific composition which is lost upon solubilization and purification of the protein