In airway smooth muscle (ASM) cells, excitation-contraction coupling is accomplished via a cascade of events that connect an elevation of cytosolic Ca²⁺concentration ([Ca²⁺]_cyt) with cross-bridge attachment and ATP-consuming mechanical work. Excitation-energy coupling is mediated by linkage of the elevation of [Ca²⁺]_cytto an increase in mitochondrial Ca²⁺concentration, which in turn stimulates ATP production. Proximity of mitochondria to the sarcoplasmic reticulum (SR) and plasma membrane is thought to be an important mechanism to facilitate mitochondrial Ca²⁺uptake. In this regard, mitochondrial movement in ASM cells may be key in establishing proximity. Mitochondria also move where ATP or Ca²⁺buffering is needed. Mitochondrial movement is mediated through interactions with the Miro-Milton molecular complex, which couples mitochondria to kinesin motors at microtubules. We examined mitochondrial movement in human ASM cells and hypothesized that, at basal [Ca²⁺]_cytlevels, mitochondrial movement is necessary to establish proximity of mitochondria to the SR and that, during the transient increase in [Ca²⁺]_cytinduced by agonist stimulation, mitochondrial movement is reduced, thereby promoting transient mitochondrial Ca²⁺uptake. We further hypothesized that airway inflammation disrupts basal mitochondrial movement via a reduction in Miro and Milton expression, thereby disrupting the ability of mitochondria to establish proximity to the SR and, thus, reducing transient mitochondrial Ca²⁺uptake during agonist activation. The reduced proximity of mitochondria to the SR may affect establishment of transient “hot spots” of higher [Ca²⁺]_cytat the sites of SR Ca²⁺release that are necessary for mitochondrial Ca²⁺uptake via the mitochondrial Ca²⁺uniporter.