The molecular mechanisms responsible for the regulation of ciliary beating frequency (CBF) are only partially characterized. To determine whether elevation of intracellular Ca²⁺ ([Ca²⁺]_i) can cause an increase in CBF, we measured CBF and Ca²⁺ in single cells. Ovine tracheal epithelial cells, obtained by dissociation with protease, were grown in primary culture for 1 to 28 days in a mucus-free system. CBF of a single cilium was measured by digital video phase-contrast microscopy and on-line Fourier-transform analysis. Changes in [Ca²⁺]_i from single cells were determined with fura-2, using ratio imaging video microscopy. Activation of a muscarinic pathway with 10 μM ACh (acetylcholine) increased [Ca²⁺]_i from 53±9 nM (mean ± s.e.m.) to 146±12 nM or to 264±51% above initial baseline. In the same cells, ACh increased CBF from a baseline of 7±0.5 Hz to 9±0.2 Hz or to 31±2.8% above baseline (n=14). The elevations of both [Ca²⁺]_i and CBF were transient and relaxed back to an elevated plateau (10/14 cells) as long as ACh was present. To elevate [Ca²⁺]_i by mechanisms independent of a G-protein-coupled receptor, we measured [Ca²⁺]_i and CBF of the same cells in extracellular solutions with either 0 Ca²⁺ (+ 1 mM EGTA) or 10 mM Ca²⁺. Both signals rose and fell with similar kinetics in response to changing [Ca²⁺]_o, suggesting that changes in [Ca²⁺]_i alone can modulate CBF. In a second independent manipulation, cells were treated with 1 μM thapsigargin, an irreversible inhibitor of the endoplasmic reticulum Ca²⁺-ATPase. Upon thapsigargin addition, 37 of 42 cells showed a transient [Ca²⁺]_i increase and, as measured in different experiments, 8 of 9 cells showed a transient increase in CBF. Interestingly, application of ACh after cells were treated with thapsigargin produced decreases in both [Ca²⁺]_i and CBF in 8/8 cells. Lastly, after 1-3 days in culture, addition of 10 μM ACh often produced [Ca²⁺]_i oscillations rather than transients in [Ca²⁺]_i. Measurements of CBF in these cells showed frequency modulation of CBF with the same peak-to-peak time interval as the Ca²⁺ oscillation. These results show that: (1) CBF can be measured from a single cilium and monitored on-line to track changes; (2) CBF and [Ca²⁺]_i can be measured in the same single cell; (3) transient changes in [Ca²⁺]_i (induced by 4 different manipulations) are associated with kinetically similar changes in CBF; and (4) [Ca²⁺]_i oscillations are coupled to frequency modulation of ciliary beating. Taken together, these results provide strong evidence that [Ca²⁺]_i is a critical intracellular messenger in the regulation of CBF in mammalian tracheal epithelial cells.