P-glycoprotein is a highly conserved membrane protein shown to be overexpressed in many multidrug-resistant tumor cell lines. P-glycoprotein is encoded by a small gene family in mammalian cells. Class I and II isoforms cause multidrug resistance, whereas class III does not. In this report, we have characterized three P-glycoprotein-specific monoclonal antibodies (mAbs) by high-resolution epitope mapping with a series of hexapeptides. mAb C494 is gene specific, binding to a sequence present only in the class I isoform of hamster and human. The mAb C32 recognizes a sequence conserved in hamster class I and II isoforms but not in class III isoforms. In contrast, the mAb C219 recognizes a highly conserved amino acid sequence found in all P-glycoprotein isoforms characterized to date. These mAbs were used to reveal differential expression and specific localization of the three P-glycoprotein isoforms in hamster tissues by immunohistochemical staining and competition with epitope-specific peptides. Colonic epithelial cells expressed predominantly the class I isoform in a polarized manner, adrenal cortical cells expressed predominantly the class II isoform, whereas a small percentage of skeletal muscle fibers expressed the class III isoform of P-glycoprotein. These findings suggest that the P-glycoprotein isoforms have distinct physiological roles associated with specialized cell functions.