Epoxyeicosatrienoic acids (EETs), products of the cytochrome P-450 monooxygenase metabolism of arachidonic acid, can regulate the activity of ion channels. We examined the effects of EETs on cardiacL-type Ca²⁺ channels that play important roles in regulating cardiac contractility, controlling heart rate, and mediating slow conduction in normal nodal cells and ischemic myocardium. Our experimental approach was to reconstitute porcineL-type Ca²⁺ channels into planar lipid bilayers where we could control the aqueous and lipid environments of the channels and the regulatory pathways that change channel properties. We found that 20 to 125 nM EETs inhibited the open probability of reconstituted L-type Ca²⁺ channels, accelerated the inactivation of the channels, and reduced the unitary current amplitude of open channels. There was no selectivity among different EET regioisomers or stereoisomers. When 11,12-EET was esterified to thesn-2 position of phosphatidylcholine, restricting it to the hydrophobic phase of the planar lipid bilayer, the reconstituted channels were similarly inhibited, suggesting that the EET interacts directly with Ca²⁺ channels through the lipid phase. The inhibitory effects of EET persisted in the presence of microcystin, an inhibitor of protein phosphatases 1 and 2A, suggesting that dephosphorylation was not the mechanism through which these eicosanoids down-regulate channel activity. This inhibition may be an important protective mechanism in the setting of cardiac ischemia where arachidonic acid levels are dramatically increased and EETs have been shown to manifest preconditioning-like effects.