1. In ovine ciliated tracheal epithelial cells, acetylcholine (ACh) activates signal transduction pathways that not only transiently increase cytoplasmic Ca2+ ([Ca2+]i) but also actively lower [Ca2+]i. The pathway for decreasing [Ca2+]i is clearly revealed after depletion of intracellular Ca2+ stores by thapsigargin (Tg), 2,5-di-(tert-butyl)-1,4-benzohydroquinone or NiCl2. Measurements with microinjected fura-2 excluded a [Ca2+] measurement artefact. 2. A four-compartment model to simulate calcium transients in non-excitable cells (consisting of a plasma membrane Ca2+ pump and channel; Ca2+ store with pump and channel; and cytosolic Ca2+ buffer) could not account for the observed [Ca2+]i decrease. We therefore explored, by simulation and experimentation, several different mechanisms that could account for it. 3. The ACh-stimulated [Ca2+]i decrease was not due to an inhibition of Ca2+ influx (Ca2+ channel blockers or absence of extracellular calcium had no effect), activation of a plasma membrane Ca2+-ATPase (two inhibitors, vanadate (30 mM) and lanthanum (10 mM), had no effect) or inhibition of the Na+-Ca2+ exchanger (replacing extracellular Na+ with N-methylglucamine had no effect). 4. The application of mitochondrial uncouplers (5 microM CCCP or 5 microM FCCP), eliminated the ACh-induced [Ca2+]i decrease. Addition of CCCP at the nadir of the decrease restored intracellular calcium levels of Tg-treated cells to baseline faster than controls not exposed to mitochondrial uncouplers. CCCP application to naïve cells did not block the ACh-induced transient increase in [Ca2+]i. 5. These data suggest that ACh-induced [Ca2+]i decreases in ciliated cells are caused by stimulated Ca2+ uptake into mitochondria.