The photoaffinity reagent 8‐azido‐α‐[32P]ATP was used to label a protein of 170 kDa in membrane vesicle preparations from a highly multidrug‐resistant cell line, KB‐V1, but not from the drug‐sensitive parental cell line KB‐3‐1. The 170‐kDa labeled protein was immunoprecipitated with a monoclonal antibody (MRK‐16) to P glycoprotein. Both ATP and GTP inhibited labeling by 8‐azido‐α‐[52P]ATP. Labeling of P170 was not inhibited by 5 mM ADP, 5 mmribose‐5‐phosphate, or 100 μmvinblastine. These data directly demonstrate that P glycoprotein has a nucleotide‐binding site that could supply energy for drug transport.— Cornwell, M. M.; Tsuruo, T; Gottesman, M. M.; Pastan, I. ATP‐binding properties of P glycoprotein from multidrug‐resistant KB cells.FASEB J1: 51‐54; 1987.