Self-association of the transactivator HBx protein of hepatitis B virus was investigated using the yeast two-hybrid system. Expression vectors for the full-length H13x (X0) and its truncated mutants (X15 and X16) were constructed by separately ligating the DNA-binding (BD) and transactivation domains (AD) of Ga14. Co-transformants of the BD and AD constructs of H13x were selected using defined minimal medium and analyzed for the reconstitution of beta-galactosidase activity. No two-hybrid interaction was observed either between the full-length H13x molecules or its highly truncated mutant X16. However, a strong functional interaction between X0 and X15, X0 and X16, and X15 and X16 suggested that HBx could self-associate in a cellular environment through its carboxy-terminal region. (C) 2004 Elsevier Inc. All rights reserved.