Many studies of Ca effects on mitochondrial respiration in intact cells have used electrical and/or chemical stimulation to elevate intracellular Ca , and have reported increases in NADH and increased ADP/ATP ratios as dominant controllers of respiration. This study tested a different form of stimulation: brief temperature increases produced by pulses of infrared light (IR, 1,863 nm, 8-10°C for ∼5 s). Fluorescence imaging techniques applied to single PC-12 cells in low µM extracellular Ca revealed IR stimulation-induced increases in both cytosolic (fluo5F) and mitochondrial (rhod2) Ca . IR stimulation increased O consumption (porphyrin fluorescence), and produced an alkaline shift in mitochondrial matrix pH (Snarf1), indicating activation of the electron transport chain (ETC). The increase in O consumption persisted in oligomycin, and began during a decrease in NADH, suggesting that the initial increase in ETC activity was not driven by increased ATP synthase activity or an increased fuel supply to ETC complex I. Imaging with two potentiometric dyes tetramethyl rhodamine methyl ester (TMRM) and R123 indicated a depolarizing shift in ΔΨ that persisted in high K medium. High-resolution fluorescence imaging disclosed large, reversible mitochondrial depolarizations that were inhibited by cyclosporin A (CSA), consistent with the opening of transient mitochondrial permeability transition pores. IR stimulation also produced a Ca -dependent increase in superoxide production (MitoSox) that was not inhibited by CSA, indicating that the increase in superoxide did not require transition pore opening. Thus, the intracellular Ca release that follows pulses of infrared light offers new insights into Ca -dependent processes controlling respiration and reactive oxygen species in intact cells. Pulses of infrared light (IR) provide a novel method for rapidly transferring Ca from the endoplasmic reticulum to mitochondria in intact cells. In PC12 cells the resulting ETC activation was not driven by increased ATP synthase activity or NADH. IR stimulation produced a Ca -dependent, reversible depolarization of ΔΨ that was partially blocked by cyclosporin A, and a Ca -dependent increase in superoxide that did not require transition pore opening.