In developing gene therapy for cystic fibrosis (CF) airways disease, a transgene encoding a partially deleted CF transmembrane conductance regulator (CFTR) Cl- channel could be of value for vectors such as adeno-associated virus that have a limited packaging capacity. Earlier studies in heterologous cells indicated that the CFTR R (regulatory) domain is predominantly random coil and that parts of the R domain can be deleted without abolishing channel function. Therefore, we designed a series of CFTR variants with shortened R domains (between residues 708 and 835) and expressed them in well-differentiated cultures of CF airway epithelia. All of the variants showed normal targeting to the apical membrane, and for the constructs we tested, biosynthesis was like wild type. Moreover, all constructs generated transepithelial Cl- current in CF epithelia. Comparison of the Cl- transport suggested that the length of the R domain, the presence of phosphorylation sites, and other factors contribute to channel activity. A variant deleting residues 708-759 complemented CF airway epithelia to the same extent as wild-type CFTR and showed no current in the absence of cAMP stimulation. In addition, expression in nasal mucosa of CF mice corrected the Cl- transport defect. These data provide insight into the structure and function of the R domain and identify regions that can be deleted with retention of function. Thus they suggest a strategy for shortening the transgene used in CF gene therapy.