1. We analysed the kinetics of coupling between cytoplasmic calcium ([Ca2+](i)) and ciliary beat frequency (CBF) using simultaneous single cilium recording and single cell [Ca2+](i) measurements from cultured ovine tracheal epithelial cells. 2. CBF and [Ca2+](i) (indicated by fura-2) were measured at rest and in response to activation of the G-protein coupled M3 muscarinic receptor by 10 μM acetylcholine (ACh). 3. Fourier transform analysis of 3 s data segments of light intensity from phase-contrast microscopy showed no significant delay between changes in [Ca2+](i) and CBF during a 2 min exposure to ACh and subsequent washout. 4. CBF time resolution was improved by computing instantaneous beat frequency. This revealed that CBF lagged the rapid increase in [Ca2+](i) in response to ACh with a delay of less than 1 beat cycle (143 ms at 7 Hz). When CBF was estimated by an improved Fourier method, this delay was observed to be 70 ± 30 ms (mean ± S.E.M.; n = 20 cilia). During the slower return to baseline, a lag of 8 ± 3.2 s was observed, indicative of hysteresis. 5. While calmodulin inhibitors (calmidazolium and W-7; each n = 5) decreased baseline CBF by an average of 1.1 ± 0.1 Hz, they did not alter the kinetic relationship between [Ca2+](i) and CBF. Similarly, phosphatase inhibitors (okadaic acid and cyclosporin A; each n = 5), changed neither baseline CBF nor the kinetic coupling between [Ca2+](i) and CBF. 6. These data suggest that the timing of Ca2+ action on CBF in ovine airway epithelial cells, is unlikely to be determined by phosphorylation reactions involving calmodulin or kinase/phosphatase reactions. 7. A simple model for Ca2+ stimulation of CBF is presented. Fits of the model to the data suggest four or more Ca2+ ions bind cooperatively to speed up CBF.
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