The UDP glucuronosyltransferases (UGT)2 are a family of enzymes which detoxify small hydrophobic compounds in mammalian cells. It is believed that UGTs are type I endoplasmic reticulum (ER) resident membrane proteins with a single membrane spanning domain near the carboxyl-terminus. The determinants of endoplasmic reticulum subcellular localization and membrane association for the UDP glucuronosyltransferase, UGT2B1, were examined. The construction and analysis of truncated and chimeric forms of UGT2B1 demonstrated that the protein contains regions of membrane interaction in the amino-terminal half of the lumenal domain in addition to the carboxyl-terminal transmembrane domain. UGT2B1 also remained resident in the ER in the absence of the cytosolic tail and transmembrane domain. Construction and analysis of an active, truncated form of UGT2B1 indicated that the cytosolically located dilysine motif, which is a putative ER membrane targeting signal, may be redundant for residency of UGT in the ER.