Polyunsaturated fatty acids (PUFAs) modulate voltage-gated K⁺ channel inactivation by an unknown site and mechanism. Effects of -6 and -3 PUFAs were investigated on the heterologously expressed K_v1.4 channel. PUFAs inhibited wild-type K_v1.4 during repetitive pulsing as a result of slowing of recovery from inactivation. In a mutant K_v1.4 channel lacking N-type inactivation, PUFAs reversibly enhanced C-type inactivation (K_D, 15-43 μM). C-type inactivation was affected by extracellular H⁺ and K⁺ as well as PUFAs, and there was an interaction among the three: the effect of PUFAs was reversed during acidosis and abolished on raising K⁺. Replacement of two positively-charged residues in the extracellular pore (H508 and K532) abolished the effects of the PUFAs (and extracellular H⁺ and K⁺) on C-type inactivation, but had no effect on the lipoelectric modulation of voltage sensor activation, suggesting two separable interaction sites/mechanisms of action of PUFAs. Charge calculations suggest that the acidic head group of the PUFAs raises the pK_a of H508 and this reduces the K⁺ occupancy of the selectivity filter, stabilising the C-type inactivated state.