Many studies have shown that polyunsaturated fatty acids from marine sources can prevent ischemia-induced cardiac arrhythmias in animals and probably humans. Kv1.4 is one of the K⁺ channels responsible for I_to and we have studied the mechanism underlying the effect of arachidonic acid (C20:4n-6; AA), eicosapentaenoic acid (C20:5n-3; EPA) and docosahexaenoic acid (C22:6n-3; DHA) on Kv1.4. The Kv1.4 channel was expressed in Xenopus oocytes and currents were recorded using the two-electrode voltage clamp technique. Wild-type Kv1.4 current was inhibited during repetitive pulsing (0.5 Hz) by AA, EPA and DHA as a result of a slowing of recovery from inactivation. For example, 100 µM AA decreased current by 82±0.5 % as a result of a decrease in the time constant of recovery from 2.25±0.14 to 4.12±0.04 s (n=5). In the wild-type channel, whereas N-type inactivation is responsible for inactivation, C-type inactivation controls recovery from inactivation. To study the effect on C-type inactivation directly, we expressed a mutant channel, which lacks N-type inactivation (fKv1.4 Δ2-146). In the mutant channel, AA, EPA and DHA dramatically enhanced the onset of C-type inactivation with a K_D of 43, 15 and 18 μM, respectively. Acidosis enhances C-type inactivation of Kv1.4, while raised extracellular K⁺ reduces it. We observed an interaction among the effects of AA, DHA, pH and K⁺ on Kv1.4: although 30 µM AA or DHA markedly enhanced C-type inactivation of Kv1.4 when extracellular pH was 7.4 and extracellular K⁺ was 3 mM, they had no effect when the pH was reduced to 5.5 or K⁺ was raised to 100 mM (n=5). Replacement of either of two positively-charged extracellular residues with a cysteine residue (H508C, K532C) abolishes the effect of extracellular pH and K⁺. The same mutations abolished the effect of polyunsaturated fatty acids. Analysis of a comparative model for the structure of the Kv1.4 channel indicates that AA could insert into the membrane just outside of the pore domain. In such a configuration, the negatively-charged headgroup of AA would interact with H508, stabilising it in the protonated state. This effect would in turn influence the selectivity filter and K⁺ ion binding, enhancing C-type inactivation. Our model is consistent with the experimental data, since the effect of AA is lost in the H508C mutant. It is concluded that application of polyunsaturated fatty acids, via the positively charged residues, H508 and K532, in the extracellular mouth of the channel, enhance C-type inactivation – this causes a slowing of recovery from inactivation and, thus, an inhibition of current during repetitive pulsing.