pHi affects a number of cellular functions, but the influence of pHi on mammalian ciliary beat frequency (CBF) is not known. CBF and pHi 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 pHi 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 pHi 7.78 +/- 0.02 resulted in a 2.2 +/- 0.1 Hz CBF increase (P < 0.05). Following removal of NH4Cl, pHi decreased to 7.24 +/- 0.02 and CBF to 5.8 +/- 0.1 Hz (P < 0.05). Removal of extracellular CO2 to change pHi resulted in similar CBF changes. Pre-activation of cAMP-dependent protein kinase (10 microM forskolin), broad inhibition of protein kinases (100 microM H-7), inhibition of PKA (10 microM H-89), nor inhibition of phosphatases (10 microM cyclosporin + 1.5 microM okadaic acid) changed pHi-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 pHi-induced CBF changes, pHi may directly act on the ciliary motile machinery.