Monoclonal antibodies (MAbs) were raised against partially purified Class I P-glycoprotein from multidrug-resistant Chinese hamster ovary CHRB30 cells. Fifteen stable monoclonal hybridoma cell lines were established, and the secreted antibodies were classified into 8 groups on the basis of banding pattern on immunoblots of P-glycoprotein digested with cyanogen bromide or partially digested with proteases. One representative of each group was tested further for several activities. Six of the 8 recognized human P-glycoprotein in the multidrug-resistant SKVLBI cell line. None of the antibodies recognized P-glycoprotein in unfixed cells, suggesting that all recognize cytoplasmic epitopes or extracellular epitopes not accessible in native P-glycoprotein. All 8 antibodies were able to immunoprecipitate P-glycoprotein from non-denaturing detergent solution. The linear epitopes of the antibodies were mapped to 11-27 amino acids. Two of the antibodies had epitopes in the linker region, 3 in the N-terminal nucleotide binding domain, 2 in the C-terminal nucleotide binding domain and 1 in the predicted cytoplasmic loop between predicted transmembrane helices 8 and 9. These antibodies, with known epitopes, could have uses for P-glycoprotein detection, structure/function studies, purification and quantitation.