Little is known about the molecular characteristics of the voltage-activated K⁺ (K_v) channels that underlie the A-type K⁺ current in vascular smooth muscle cells of the systemic circulation. We investigated the molecular identity of the A-type K⁺ current in retinal arteriolar myocytes using patch-clamp techniques, RT-PCR, immunohistochemistry, and neutralizing antibody studies. The A-type K⁺ current was resistant to the actions of specific inhibitors for K_v3 and K_v4 channels but was blocked by the K_v1 antagonist correolide. No effects were observed with pharmacological agents against K_v1.1/2/3/6 and 7 channels, but the current was partially blocked by riluzole, a K_v1.4 and K_v1.5 inhibitor. The current was not altered by the removal of extracellular K⁺ but was abolished by flecainide, indicative of K_v1.5 rather than K_v1.4 channels. Transcripts encoding K_v1.5 and not K_v1.4 were identified in freshly isolated retinal arterioles. Immunofluorescence labeling confirmed a lack of K_v1.4 expression and revealed K_v1.5 to be localized to the plasma membrane of the arteriolar smooth muscle cells. Anti-K_v1.5 antibody applied intracellularly inhibited the A-type K⁺ current, whereas anti-K_v1.4 antibody had no effect. Co-expression of K_v1.5 with K_vβ1 or K_vβ3 accessory subunits is known to transform K_v1.5 currents from delayed rectifers into A-type currents. K_vβ1 mRNA expression was detected in retinal arterioles, but K_vβ3 was not observed. K_vβ1 immunofluorescence was detected on the plasma membrane of retinal arteriolar myocytes. The findings of this study suggest that K_v1.5, most likely co-assembled with K_vβ1 subunits, comprises a major component underlying the A-type K⁺ current in retinal arteriolar smooth muscle cells.