Membrane mimetic environments formed from detergents or short-chain phospholipids are widely utilized in structural studies of membrane proteins. Using site-directed spin labeling (SDSL), we show that micelle and isotropic bicellar environments alter the N-terminal region of BtuB, the outer membrane vitamin B12 transporter found in Escherichia coli. These membrane mimetic systems promote an unfolding of the N-terminus of the protein that does not occur when the protein is in either native or reconstituted bilayers. The N-terminal Ton box of BtuB has been shown to exist in two conformations, depending upon the presence or absence of substrate. However, the detergent-destabilized conformation is different from either the substrate-free or the substrate-bound form of this transporter. This example demonstrates that membrane mimetic systems will not always substitute for the lamellar bilayer environment provided by a biological membrane.