Understanding membrane protein folding and stability is required for a molecular explanation of function and for the development of interventions in membrane protein folding diseases. Stable aqueous detergent solutions of the Escherichia coli glycerol facilitator in its native oligomeric state have been difficult to prepare as the protein readily unfolds and forms nonspecific aggregates. Here, we report a study of the structure and stability of the glycerol facilitator in several detergent solutions by Blue Native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD), and fluorescence. Protein tetramers were prepared in neutral dodecyl maltoside (DDM) and in zwitterionic lysomyristoylphosphatidylcholine (LMPC) detergent solutions that are stable during SDS-PAGE. Thermal unfolding experiments show that the protein is more stable in LMPC than in DDM. Tertiary structure unfolds before quaternary and some secondary structure in LMPC, whereas unfolding is more cooperative in DDM. The high stability of the protein in DDM is evident from the unfolding half-life of 8 days in 8 M urea, suggesting that hydrophobic interactions contribute to the stability. The protein unfolds readily in LMPC below pH 6, whereas the tetramer remains intact at pH 4 in DDM. At pH 4 in DDM, the protein is more sensitive than at neutral pH to unfolding by SDS and the effect is reversible. At pH 3 in DDM, the tetramer unfolds, losing its tertiary structure but retaining native helical structure which melts at significantly lower temperatures than in the native tetramer. The glycerol facilitator prepared in SDS is mainly monomeric and has ~10% less alpha-helix than the native protein. CD suggests that it forms a condensed structure with non-native tertiary contacts highly similar to the state observed in LMPC at low pH. The implications of the results for in vitro and in vivo folding of the protein are discussed.