Each homologous half of P-glycoprotein consists of a transmembrane domain with six potential transmembrane segments and an ATP-binding domain. Labeling studies with photoactive drug analogs show that labeling occurs within or close to predicted transmembrane segments (TM) 6 (residues 331-351) and TM12 (residues 974-994). To test if these segments are in near-proximity we generated 42 different P-glycoprotein mutants in which we re-introduced a pair of cysteine residues into a Cys-less P-glycoprotein, one within TM6 (residues 332-338) and one within TM12 (residues 975-980) and assayed for cross-linking between the cysteines. All the mutants retained verapamil-stimulated ATPase activity. We found that only the mutant containing Cys-332 and Cys-975 was cross-linked in the presence of oxidant as judged by its decreased mobility on SDS gels. Similar results were obtained when the same mutations were introduced into Cys-less NH2-terminal and COOH-terminal half-molecules of P-glycoprotein followed by coexpression and treatment with oxidant. Cross-linking between Cys-332 and Cys-975, however, was inhibited by verapamil or vinblastine but not by colchicine. These results suggest that residues Cys-332 and Cys-975, which occupy equivalent positions when TM6 and TM12 are aligned, are close to each other in the tertiary structure of P-glycoprotein.