EmrD is the only structurally characterized drug/H(+) antiporter of the major facilitator superfamily (MFS). It has been crystallized in a doubly occluded conformation that is considered representative of an intermediate state in the transport cycle of MFS transporters. However, unexpected features of the crystal structure and the lack of functional information available for EmrD limit the utility of the structural data. To assess whether the crystal structure represents a stable state in a native-like environment, we used electron paramagnetic resonance (EPR) spectroscopy to determine the mobility and accessibility of spin labels at 76 positions in six transmembrane (TM) helices of EmrD reconstituted in liposomes. While the EPR data were mostly consistent with the crystal structure, they also revealed significant deviations from the predicted orientation and topology of TM helices at several locations. Additionally, we were unable to reproduce EmrD-dependent multidrug resistance phenotypes in vitro and in cell-based assays of drug transport. In spite of structural and functional discrepancies, we mapped a pH-dependent conformational change in which the cytoplasmic side of the N-terminal half opened locally in response to protonation. This conformational switch is consistent with the expected pH-dependent behavior of MFS H(+)-coupled antiporters.