Kv1.4 is known to encode a transient outward K⁺ current in the heart, including in the human. The ω-3 polyunsaturated fatty acids, eicosapentaenoic acid (C20:5n-3; EPA) and docosahexaenoic acid (C22:6n-3; DHA), found in fish oils, are known to play a protective role against the development of ventricular fibrillation accompanying myocardial ischaemia. On the other hand, there is known to be an accumulation of the ω-6 polyunsaturated fatty acid, arachidonic acid (C20:4n-6; AA) in the ischaemic myocardium (there can be a >3-fold increase in free AA to ~30 µM). We have studied the effect of AA, EPA and DHA on Kv1.4. A mutant Kv1.4 channel that lacks N-type inactivation (fKv1.4 Δ2-146) was expressed in Xenopus oocytes, and currents were recorded using the two-electrode voltage clamp technique. AA, EPA and DHA dramatically enhanced fKv1.4 Δ2–146 C-type inactivation with a K_D of 43, 15 and 18 μM, respectively (n=10, 7 and 5). Replacement of either of two positively-charged pore residues with a cysteine residue (H508C; K532C) abolished the enhancement caused by AA and EPA (n = 5 and 5). It is known that these mutations cause a loss of sensitivity of Kv1.4 to pH and extracellular K⁺ (pH and extracellular K⁺ also affect C-type inactivation of Kv1.4). We observed an interaction among the effects of AA, pH and [K⁺]_o: in the presence of 30 µM AA, the effect of pH on C-type inactivation of Kv1.4 was markedly reduced (n = 5), while raising extracellular K⁺ abolished the effect of AA on C-type inactivation (n = 5). We conclude that AA, DHA and EPA cause a dose-dependent enhancement of C-type inactivation of Kv1.4 via a mechanism it shares in common with extracellular pH and K⁺ and involving extracellular positively-charged pore residues. This inhibition of Kv1.4 by polyunsaturated fatty acids may involve a binding of the negatively charged polyunsaturated fatty acids to the positively charged residues (H508 and K532) in the extracellular mouth of the channel.