pH_i affects a number of cellular functions, but the influence of pH_i on mammalian ciliary beat frequency (CBF) is not known. CBF and pH_i of single human tracheobronchial epithelial cells in submerged culture were measured simultaneously using video microscopy (for CBF) and epifluorescence microscopy with the pH‐sensitive dye BCECF. Baseline CBF and pH_i values in bicarbonate‐free medium were 7.2 ± 0.2 Hz and 7.49 ± 0.02, respectively (n= 63). Alkalization by ammonium pre‐pulse to pH_i 7.78 ± 0.02 resulted in a 2.2 ± 0.1 Hz CBF increase (P < 0.05). Following removal of NH₄Cl, pH_i decreased to 7.24 ± 0.02 and CBF to 5.8 ± 0.1 Hz (P < 0.05). Removal of extracellular CO₂ to change pH_i resulted in similar CBF changes. Pre‐activation of cAMP‐dependent protein kinase (10 μm forskolin), broad inhibition of protein kinases (100 μm H‐7), inhibition of PKA (10 μm H‐89), nor inhibition of phosphatases (10 μm cyclosporin + 1.5 μm okadaic acid) changed pH_i‐mediated changes in CBF, nor were they due to [Ca²⁺]_i changes. CBF of basolaterally permeabilized human tracheobronchial cells, re‐differentiated at the air–liquid interface, was 3.9 ± 0.3, 5.7 ± 0.4, 7.0 ± 0.3 and 7.3 ± 0.3 Hz at basolateral i.e., intracellular pH of 6.8, 7.2, 7.6 and 8.0, respectively (n= 18). Thus, intracellular alkalization stimulates, while intracellular acidification attenuates human airway CBF. Since phosphorylation and [Ca²⁺]_i changes did not seem to mediate pH_i‐induced CBF changes, pH_i may directly act on the ciliary motile machinery.