P-glycoprotein (Pgp)1 is a polytopic membrane protein and functions as an energy-dependent drug efflux pump. It is responsible for multidrug resistance (MDR) in cancer cell lines. Recently, the topological structure of Pgp has been investigated. However, the results are in dispute. A major question concerning the Pgp topology is the membrane orientation of the loop linking TM4 and TM5 (loop 4) and the loop linking TM8 and TM9 (loop 8). In this study, we generated polyclonal antibodies specific to these two loops. In combination with a panel of other well-characterized site-specific polyclonal- and monoclonal antibodies of Pgp, we tested the membrane orientation of these two loops of Pgp in multidrug-resistant cells using immunocytochemistry and proteolysis/membrane protection assay. Our results showed that (1) both loops 4 and 8 are located extracellularly whereas other domains, such as the ATP-binding sites, are in the cytoplasm and (2) proteolysis of Pgp is not a random event and the trypsin-sensitive sites are cleaved in orders. Since the Pgp was not genetically manipulated in this study, in contrast to previous studies, we believe that naturally expressed Pgp molecules have an unconventional topology. We speculate that this alternate topology of Pgp may represent a different functional state of the protein. Further detailed analysis of Pgp topology will help to understand the fundamental mechanism of drug transport by Pgp.