To better understand the mechanisms by which PKA-dependent phosphorylation regulates CFTR channel activity, we have assayed open probabilities (P_o), mean open time and mean closed time for a series of CFTR constructs with mutations at PKA phosphorylation sites in the R-domain. Forskolin-stimulated channel activity was recorded in cell-attached and inside-out excised patches from transiently transfected CHO cells. Wild type CFTR and constructs with a single S-to-A mutation as well as octa (S-to-A mutations at 8 sites) and constructs with 1 or 2 A-to-S mutations were studied. In cell-attached patches, S-to-A mutations at amino acids 700, 795 or 813 decreased P_o, whereas S-to-A mutations at 737 or 768 increased P_o. In general, differences in P_o were due to differences in mean closed time. For selected constructs with both high and low values of P_o channel activity was measured in excised patches. With 1 mM ATP P_o values were similar to those observed in cell-attached patches, but with 10 mM ATP all constructs tested showed elevated values of P_o. ATP-dependent increases in P_o were due to reductions in mean closed times. These results indicate that R-domain phosphorylation affects ATP binding and not the subsequent steps of hydrolysis and channel opening. A model is developed where R-domain phosphorylation, in a site-dependent manner, alters an equilibrium between forms of CFTR with low and high affinities for ATP.