Phosphorylation of P-glycoprotein (Pgp) by protein kinase C occurs on apparently the same sites in vitro and in intact cells (in situ) and is implicated in modulation of Pgp function. The region of the molecule which contains the in vitro phosphorylation sites and two specific sites within this region are now determined by peptide sequencing. Membrane vesicles from multidrug-resistant human KB-V1 cells were incubated with purified protein kinase C and [gamma-32P]ATP, and Pgp (containing 1 mol of phosphate/mol of protein) was purified to apparent homogeneity. Phosphorylation occurred exclusively on serine residues. Phosphopeptides were generated by digestion with Lys-C endoproteinase or trypsin, partially purified by high performance liquid chromatography, and further purified with strategies developed for individual phosphopeptides. Sequence analysis by Edman degradation and comparison with the deduced amino acid sequence of human (mdr 1) Pgp identified serines 661 and 671, and one or more of serines 667, 675, and 683, as sites of phosphorylation. These sites are clustered in the linker region located between the two homologous halves of Pgp. Our results identify a previously undefined, phosphorylatable domain of Pgp, smaller in size but analogous in location to the R-domain of the cystic fibrosis transmembrane conductance regulator. These data provide a basis for a better understanding of the role of phosphorylation in the mechanism of action and regulation of this important multidrug pump protein.