Hepatitis B surface antigen (HBsAg) is the major component of the envelope of hepatitis B virus (HBV). As a resident membrane protein in the endoplasmic reticulum, it plays a key role in the viral morphogenesis. Little is known about cellular proteins that interact with HBsAg and thereby contributing to HBV morphogenesis. Using the yeast split-ubiquitin system, a number of cellular membrane proteins have been isolated in this study. These include a resident protein of endoplasmic reticulum (thioredoxin-related transmembrane protein 2), an adaptor protein involved in clathrin-mediated endocytosis and HIV-mediated downregulation of CD4, and a co-receptor of coxsakie B virus. The significance of our findings is suggested by the identification of cellular membrane proteins interacting with other virus proteins. Further functional analysis of these HBsAg- interacting cellular membrane proteins should shed new insights on their role in HBV morphogenesis.