Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel activated by PKA-dependent phosphorylation on its regulatory domain (R domain). Previous studies have demonstrated that the R domain controls phosphorylation-dependent CFTR activation by multiple effects including inhibition and stimulation on channel activity. However, it is unclear whether specific regions in the R domain are account for these mechanisms controlling CFTR activation. To test this hypothesis by screening the function of different R domain regions, we constructed a variety of R domain deletions over the region from residue 708 to 835 in CFTR. We studied the single-channel activity and PKA-independent constitutive Cl- currents of these CFTR constructs. Our data indicate that deleting region 784-835 generated little constitutive Cl – currents in the presence of 1 mM ATP alone, but reduced greatly CFTR channel activity after PKA phosphorylation. Further studies on small deletions in this region suggest that whole region 784-835 regulates the closing rate of the CFTR channel, whereas region 829-835 also modulates the channel opening rate. Therefore, the C-terminal part of the R domain is required for normal CFTR activity. Conversely, deletions of region 708-759 and region 760-783 had small effects on channel activity. Moreover, deleting region 760-783 or subregions 760-769 and 770-776 produced large constitutive Cl- currents. These data suggest that region 760-776 may form an inhibitory motif that prevents CFTR activation. Taken together, our data suggest that the small R domain regions in different locations may form functional motifs that either inhibit or stimulate the channel activity of CFTR.