The myogenic response of cerebral arteries is critical for regulation of blood flow to the brain, maintaining constant flow during variations in systemic blood pressure via graded changes in diameter. Precisely controlled changes in membrane potential are involved, but the underlying ionic mechanisms are not completely defined. A role for voltage-gated K⁺ (Kv) channels was indicated using 4-aminopyridine (Knot & Nelson, 1995) and Kv1 family subunits were shown to be expressed by cerebral arteries and arterioles (Cheong et al. 2001; Albarwani et al. 2003). This study employed a dominant-negative, myc-tagged rabbit Kv1.5 (Kv1.5DN) pore-mutant subunit to assess the contribution of Kv1 channels to the myogenic response of rat middle cerebral arteries. The Kv1.5DN construct was first shown to reduce whole-cell currents due to wild-type rabbit Kv1.5 (Kv1.5WT) and Kv1.2, but not Kv2 channels expressed in HEK293 cells. Expression and co-assembly of Kv1.5DN with wild-type Kv1 was confirmed by co-immunoprecipitation. Myogenic reactivity of freshly isolated arteries, as well as arteries cultured for 48 hrs after transfection (Opazo-Saez et al.,2004) with empty plasmid (pcDNA3), Kv1.5DN or Kv1.5WT was assessed by pressure myography (Knot & Nelson, 1995). Rats were humanely killed. Expression of Kv1.5DN enhanced, whereas Kv1.5WT suppressed myogenic reactivity compared to arteries transfected with empty plasmid. The myogenic responses of empty plasmid, Kv1.5DN and Kv1.5WT vessels at 80 mmHg (i.e. difference in diameter in Ca²⁺-containing vs. Ca²⁺-free solutions) were 79.3±5.3 μm (mean±S.E.M.; n=11), 109.6±7.3 μm (n=9) and 56.9±4.0 μm (n=9), respectively (P<0.05 by ANOVA). There was no difference between the response of freshly isolated (89.4±10.3 μm; n=5) and cultured, empty plasmid vessels. Message encoding rabbit Kv1.5DN or Kv1.5WT was identified using rabbit Kv1.5-specific primers in RNA samples of transfected, but not freshly isolated or empty plasmid rat arteries by RT-PCR. All amplicons were sequenced to confirm their identity. This study provides the first molecular evidence for a contribution of Kv1 channels to the control of myogenic reactivity of resistance arteries. Kv1 channel activation in response to myogenic depolarization likely serves as a negative-feedback mechanism, providing for precise control over the extent of L-type Ca²⁺ channel activation and arterial constriction evoked by increased intraluminal pressure.