Monotopic membrane proteins integrate into the lipid bilayer via reentrant hydrophobic domains that enter and exit on a single face of the membrane. Whereas many membrane-spanning proteins have been structurally characterized and transmembrane topologies can be predicted computationally, relatively little is known about the determinants of membrane topology in monotopic proteins. Recently, we reported the X-ray structure determination of PglC, a full-length monotopic membrane protein with phosphoglycosyl transferase (PGT) activity. The definition of this unique structure has prompted in vivo, biochemical, and computational analyses to understand and define key motifs that contribute to the membrane topology and to provide insight into the dynamics of the enzyme in a lipid bilayer environment. Using the new information gained from studies on the PGT superfamily we demonstrate that two motifs exemplify principles of topology determination that can be applied to the identification of reentrant domains among diverse monotopic proteins of interest.