P-glycoprotein (P-gp) is thought to function as a drug efflux pump in multidrug resistant (MDR) cells. The functional form of P-gp in its native state is not known. Previous results from radiation target size analysis have suggested that P-gp occurs as dimers in MDR cell plasma membranes [Boscoboinik et al. (1990) Biochim. Biophys. Acta 1027, 225-228]. In this study, we used sucrose gradient velocity sedimentation to determine if P-gp oligomers could be retrieved from detergent extracts of hamster and human MDR cell lines. The proportion of P-gp recovered as higher order oligomers was dependent on the detergents used for solubilization of the cells. When a detergent such as CHAPS was used, 50% or more of the P-gp sedimented as higher order oligomers. In contrast, in the presence of SDS, only monomers were retrieved, but naturally occurring oligomers could be preserved if the cells were treated with a cross-linker prior to detergent solubilization. The oligomers and monomers were both able to bind the photoactive analog of ATP (8-azido[alpha-32P]ATP) or the drug [3H]azidopine in membrane preparations. P-gp is a phosphoprotein, and its phosphorylated state is thought to be important for function. When MDR cells were labeled with [32P]orthophosphate in vivo, we observed that the monomer and dimer were more highly phosphorylated than the larger oligomers, suggesting that these different forms of P-gp may be functionally distinct. The assembly of oligomers appears to occur in an early bisynthetic compartment, and asparagine-linked glycosylation is not required for their formation. Our findings indicate that oligomers of P-gp exist in MDR cells and raise the possibility that the dynamics of oligomer formation and dissociation may be important in the mechanism of action of P-gp.