The antimycotic drug caspofungin is known to alter the cell function of cardiomyocytes and the ciliabearing cells of the tracheal epithelium. The objective of this study was to investigate the homeostasis of intracellular ca 2+ concentration ([ca 2+ ] i) after exposure to caspofungin in isolated human tracheal epithelial cells. The [Ca 2+ ] i was measured using the ratiometric fluoroprobe FURA-2 AM. We recorded two groups of epithelial cells with distinct responses to caspofungin exposure, which demonstrated either a rapid transient rise in [ca 2+ ] i or a sustained elevation of [ca 2+ ] i. Both patterns of Ca 2+ kinetics were still observed when an influx of transmembraneous Ca 2+ ions was pharmacologically inhibited. Furthermore, in extracellular buffer solutions without Ca 2+ ions, caspofungin exposure still evoked this characteristic rise in [ca 2+ ] i. To shed light on the origin of the Ca 2+ ions responsible for the elevation in [ca 2+ ] i we investigated the possible intracellular storage of ca 2+ ions. The depletion of mitochondrial ca 2+ stores using 25 µM 2,4-dinitrophenol (DNP) did not prevent the caspofungin-induced rise in [ca 2+ ] i , which was rapid and transient. However, the application of caffeine (30 mM) to discharge Ca 2+ ions that were presumably stored in the endoplasmic reticulum (eR) prior to caspofungin exposure completely inhibited the caspofungin-induced changes in [ca 2+ ] i levels. When the ER-bound IP 3 receptors were blocked by 2-APB (40 µM), we observed a delayed transient rise in [Ca 2+ ] i as a response to the caspofungin. Inhibition of the ryanodine receptors (RyR) using 40 µM ryanodine completely prevented the caspofungin-induced elevation of [ca 2+ ] i. In summary, caspofungin has been shown to trigger an increase in [ca 2+ ] i independent from extracellular ca 2+ ions by liberating the ca 2+ ions stored in the ER, mainly via a RyR pathway. The mucociliary clearance of lower airways contributes to the removal of debris and pathogens that are trapped in the upper mucus layer from the lower airways. This complex mechanism is driven by cilia-bearing cells of the bronchiolar and tracheal epithelium. The kinocilia of these cells bear over 200 motor proteins as a propelling apparatus, including dynein, the ATP hydrolyzing enzyme 1,2. Under basal conditions, cilia beat continuously without external stimulation but can beat faster when necessary. This elevated beating frequency depends on several interdepending signal transduction cascades, including changes in intracellular concentrations of Ca 2+ ions ([Ca 2+ ] i) 3-6. However, most signal cascades in these cells depend on at least a transient [Ca 2+ ] i to be activated in order to allow the cilia to beat faster for prolonged periods 7,8. Thus, the regulation of [Ca 2+ ] i is a cornerstone for many cellular signal cascades and cell functions. In epithelial cells in the airway, different canonical Ca 2+ stores or Ca 2+ influx pathways are involved in the regulation of [Ca 2+ ] i. The known intracellular stores are the