1. The effects of the sequential stimulation of ciliary beat frequency (CBF) via two different phosphorylation cascades (dependent on protein kinase A (PKA) and calmodulin, respectively) were determined using video microscopy applied to a perfused preparation of human nasal respiratory epithelium in vitro. Dibutyryl cyclic AMP (db-cAMP) (10(-3) M) was used to stimulate PKA and the calcium ionophore 4-Br-A23187 (10(-5) M) was used to stimulate calmodulin-dependent phosphorylation. 2. Perfusion with db-cAMP (10(-3) M) alone showed an early rise in CBF (15.0 +/- 4%, mean +/- S.E.M., P < 0.05) by 10 min which remained elevated for 35 min; in contrast, the highest CBF response to 4-Br-A23187 (10(-5) M) alone was not achieved until 35 min (16.1 +/- 1.8%, P < 0.05). 3. When a db-cAMP stimulus was applied to cells which had been pre-incubated with 4-Br-A23187 for 30 min, a further rise in CBF (maximal at 20 min, 14.3 +/- 2%, P < 0.05) was observed. Reversing the sequence of perfusions, cells pre-incubated with db-cAMP showed no further rise in response to stimulation with 4-Br-A23187. 4. We hypothesized that PKA inhibited the response to the 4-Br-A23187. This notion was supported by the restoration of the CBF response (22.8 +/- 4%, P < 0.05) to 4-Br-A23187 when the cells were pre-incubated with the protein kinase inhibitor 1-(5-isoquinolinyl-sulphonyl)-2-methylpiperazine (10(-3) M), before the sequential perfusions with db-cAMP and 4-Br-A23187. We conclude that the A23187-dependent pathway, which regulates intrinsic CBF, is inhibited by db-cAMP but not vice versa.