P-Glycoprotein (Pgp), an energy-dependent drug efflux pump responsible for multidrug resistance of many cancer cells, is comprised of two homologous halves connected by a peptide segment approximately 75 amino acids (aa) in length. The effects of length and composition of this connecting region on Pgp cell surface expression and the ability of the two halves to interact were explored using both stable transfections of Pgp mutants in mammalian cell lines and a vaccinia virus transient expression system. A 17 aa insertion of predicted flexible structure between amino acids 681 and 682 resulted in a functional Pgp molecule that was capable of conferring drug resistance. In contrast, an 18 aa peptide insertion with a predicted alpha-helical structure was unstable when expressed transiently. A 34 aa deletion from the central core of the linker region (Delta653-686) resulted in a protein expressed at the cell surface in amounts comparable to that of wild-type Pgp but unable to confer drug resistance. No apparent differences in drug or [alpha-32P]-8-azido-ATP photoaffinity labeling were observed. However, both ATP hydrolysis and drug transport activities of the deletion mutant were completely abrogated, indicating that the linker deletion disconnected substrate binding from ATP hydrolysis and transport. This mutant also failed to exhibit an ATP hydrolysis-dependent enhancement of binding of a conformation-sensitive monoclonal antibody, UIC2. Upon replacement with a 17 aa linker peptide having a predicted flexible secondary structure, but bearing no homology to the deleted 34 aa segment, normal Pgp transport and basal and drug-stimulated ATPase activities were restored along with increased UIC2 binding in the presence of substrate, suggesting a dramatic conformational change between the nonfunctional and functional molecules. Taken together, these data suggest a flexible secondary structure of the connector region is sufficient for the coordinate functioning of the two halves of Pgp, likely specifically required for the proper interaction of the two ATP binding sites.