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 (P_o). After a purification protocol involving wheat-germ agglutinin affinity and anion-exchange chromatography, the proteins were sequentially inserted into liposomes, which were then fused into PLBs. Functional and biochemical characterization tests confirm that the Cl channel is a 55-kDa glycosylated monomer with voltage- and Ca²⁺ concentration-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 50% inhibitory concentration 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 P_o of the native channel with respective 50% inhibitory concentration values of 0.27 and 0.30 µM but failed to alter the P_o 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 P_o requires a lipid environment of specific composition that is lost on solubilization and purification of the protein.