1. Simultaneous measurements of average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were carried out on ciliated rabbit tracheal cells in order to determine quantitatively the role of calcium in the regulation of mucus-transporting cilia. 2. Extracellular ATP caused a rapid increase in both [Ca2+]i and CBF in the 0.1-1000 microM concentration range. The rise in [Ca2+]i levelled off to an elevated [Ca2+]i plateau while the cilia remained in a high activation state. The magnitude of the rise in [Ca2+]i and CBF as well as the value of the elevated [Ca2+]i plateau and the value of the sustained CBF were dependent on the concentration of ATP in the solution. 3. No correlation was found between the mean values of [Ca2+]i and CBF at rest but a sigmoidal relationship was found to exist between the maximal rises of these parameters following excitation with extracellular ATP. This sigmoidal correlation incorporated the experiments where [Ca2+]i rise was induced by depletion of internal calcium stores with thapsigargin or by entry of calcium induced by ionomycin. 4. Extracellular ATP caused both the release of calcium from internal stores and calcium influx from the extracellular solution. The release of calcium was identified as originating from a thapsigargin-sensitive and a thapsigargin-insensitive calcium store. It is suggested that the release of calcium from these stores induces the initial rise in CBF. 5. The sustained activation of the cilia and elevated calcium plateau were found to be the result of the extracellular ATP-induced calcium influx. This calcium influx was insensitive to the voltage-gated calcium channel inhibitors verapamil and diltiazem, but was completely eliminated by lowering the extracellular calcium concentration to 0.1 microM. 6. We propose that the initial jump in the CBF is mediated by the calcium released from a thapsigargin-insensitive calcium store adjacent to the cilia, while the later, and longer, rise in CBF is the result of the calcium emanating from the thapsigargin-sensitive store which is positioned further away from the cilia within the cell cytoplasm. The calcium influx that follows is responsible for sustaining the cilia at a high level of excitation.