In smooth muscles, Ca²⁺ sparks occur as spontaneous Ca²⁺ release from superficial sarcoplasmic reticulum through ryanodine receptors (RyRs). Ca²⁺ sparks activate large-conductance Ca²⁺-activated K⁺ (BK) channels nearby and elicit spontaneous transient outward currents (STOCs), which cause the decrease in muscle tone via membrane hyperpolarization. In the present study, the imaging of molecular assembly in Ca²⁺ microdomain was obtained using confocal and total internal reflection fluorescence (TIRF) microscopes with fluorescence-labeled molecules in vascular smooth muscle cells. Under whole-cell patch-clamp, Ca²⁺ sparks in a TIRF zone elicited STOCs and consequent membrane hyperpolarization. Clusters of RyR, BK channel, and caveolin 1 (Cav1) were densely distributed within Ca²⁺ spark microdomains. FRET analyses suggest the direct molecular interaction of BK channel with Cav1. In addition, a part of voltage-dependent Ca²⁺ channels (VDCCs) were associated with BK channels and localized around Ca²⁺ spark sites. A large part of co-localized BK channel and VDCC demonstrated FRET interaction in vascular myocytes. The interaction of these channels was significantly reduced in myocytes isolated from Cav1 knockout mice, genetically lacking caveolae. These results suggest that a substantial part of BK channels in vascular myocytes compose molecular complex with VDCCs and accumulate in caveolae, where functional Ca²⁺ microdomains are supposed to be formed. The Ca²⁺ influx through VDCC may directly activate associated BK channel in a caveola, while a Ca²⁺ spark may activate more BK channels in a larger area with loose coupling. Spatiotemporal imaging of molecular assembly and Ca²⁺ dynamics in Ca²⁺ microdomain including caveolae enables us to elucidate its physiological impact in the regulation of membrane excitability and muscle tone in vascular smooth muscle cells.