Abstract: Background: The reported ability of omega-3 PUFA to improve cardiovascular function is associated with amelioration of endothelial function. Because the latter is largely controlled by endothelial electrical signaling, the impact and underlying mechanisms of action of docosahexaenoic acid (DHA), a main component of fish oil, on endothelial electrical responses was explored. Methods: Patch-clamp registration of the membrane potential, whole-cell and single currents in endothelial cell line derived from human umbilical vein. Results: In whole-cell configuration, DHA (3 μM) produced membrane hyperpolarization and stimulation of paxilline-sensitive and GDP S-insensitive outwardly-rectifying current. In inside-out configuration, DHA stimulated single channel BK activity without changing the single channel amplitude. The effect of DHA was absent after depletion of the membrane cholesterol level by preincubation with methylcyclodextrin. Conclusion: Membrane cholesterol is an essential component in BK channel sensitivity to DHA. Alterations in cholesterol concentrations within individual lipid rafts may represent a potential mechanism by which omega-3 PUFA affect cellular signaling and function via modulation of BK channels.