In airway smooth muscle (ASM) cells, excitation-contraction coupling is accomplished via a cascade of events which connect an elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) with cross-bridge attachment and ATP-consuming mechanical work. Excitation-energy coupling is mediated by linking the elevation in [Ca²⁺]_cyt to an increase in mitochondrial Ca²⁺ ([Ca²⁺]_mito) which in turn stimulates ATP production. Mitochondria proximity with 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 may be key in establishing proximity. Mitochondria also move where ATP or Ca²⁺ buffering is needed. Mitochondrial movement is mediated through interactions with the molecular complex of Miro and Milton, which couples mitochondria to kinesin motors at microtubules. We examined mitochondria movement in human ASM and hypothesized that at basal [Ca²⁺]_cyt levels, mitochondrial movement is necessary to establish proximity of mitochondria with the SR, and that during the transient increase in [Ca²⁺]_cyt induced 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⁺]_cyt at the sites of SR Ca²⁺ release that are necessary for mitochondrial Ca²⁺ uptake via the mitochondrial Ca²⁺ uniporter.