Spatiotemporal changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_c) trigger a number of physiological functions in smooth muscle cells (SMCs). We previously imaged Ca²⁺-induced Ca²⁺ release following membrane depolarization as local Ca²⁺ transients, Ca²⁺ hotspots, in subplasmalemmal regions. In this study, the physiological significance of mitochondria on local Ca²⁺ signaling was examined. Cytosolic and mitochondrial Ca²⁺ images following depolarization or action potentials were recorded in single SMCs from the guinea pig urinary bladder using a fast-scanning confocal fluorescent microscope. Depolarization- and action potential-induced [Ca²⁺]_c transients occurred at several discrete sites in subplasmalemmal regions, peaked within 30 ms, and then spread throughout the whole-cell. In contrast, Ca²⁺ concentration in the mitochondria matrix ([Ca²⁺]_m) increased after a delay of ~50 ms from the start of depolarization, and then peaked within 500 ms. Following repolarization, [Ca²⁺]_c returned to the resting level with a half decay time of ~500 ms, while [Ca²⁺]_m recovered more slowly (~1.5 s). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, a mitochondrial uncoupler, abolished depolarization-induced [Ca²⁺]_m elevations and slowed [Ca²⁺]_c changes. Importantly, short depolarization-induced changes in [Ca²⁺]_m and transmembrane potential in mitochondria coupled to Ca²⁺ hotspots were significantly larger than those in other mitochondria. Total internal reflection fluorescence imaging revealed that a subset of mitochondria closely localized with ryanodine receptors and voltage-dependent Ca²⁺ channels. These results indicate that particular mitochondria are functionally coupled to ion channels and sarcoplasmic reticulum fragments within the local Ca²⁺ microdomain, and thus, strongly contribute to [Ca²⁺]_c regulation in SMCs.