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Abstract: Nerve and muscle action potential repolarization are produced and modulated by the regulated expression and activity of several types of voltage-gated K+ (Kv) channels. Here, we show that sialylated N-glycans uniquely impact gating of a mammalian Shaker family Kv channel isoform, Kv1.5, but have no effect on gating of a second Shaker isoform, Kv1.4. Each isoform contains one potential N-glycosylation site located along the S1–S2 linker; immunoblot analyses verified that Kv1.4 and Kv1.5 were N-glycosylated. The conductance–voltage (G–V) relationships and channel activation rates for two glycosylation-site deficient Kv1.5 mutants, Kv1.5N290Q and Kv1.5S292A, and for wild-type Kv1.5 expressed under conditions of reduced sialylation, were each shifted linearly by a depolarizing ∼18 mV compared to wild-type Kv1.5 activation. External divalent cation screening experiments suggested that Kv1.5 sialic acids contribute to an external surface potential that modulates Kv1.5 activation. Channel availability was unaffected by changes in Kv1.5 glycosylation or sialylation. The data indicate that sialic acid residues attached to N-glycans act through electrostatic mechanisms to modulate Kv1.5 activation. The sialic acids fully account for effects of N-glycans on Kv1.5 gating. Conversely, Kv1.4 gating was unaffected by changes in channel sialylation or following mutagenesis to remove the N-glycosylation site. Each phenomenon is unique for Kv1 channel isoforms, indicating that sialylated N-glycans modulate gating of homologous Kv1 channels through isoform-specific mechanisms. Such modulation is relevant to changes in action potential repolarization that occur as ion channel expression and glycosylation are regulated. [Copyright &y& Elsevier]