Expression of the gene encoding the hepatitis B virus middle surface antigen (pre-HBsAg) in the yeast Yarrowia lipolytica has been studied. The preS2-HBsAg gene was expressed from the alkaline extracellular protease-encoding gene (XPR2) promoter. In the fusion construct, the membrane-spanning 'a' domain of preS2-HBsAg has been replaced by the leader peptide and the proI region of the alkaline protease, thus eliminating the epitope responsible for the immune escape mechanism. Expression has been found to be growth-stage dependent with the highest protein accumulation during the stationary phase, accounting for around 2.35% of the total soluble intracellular proteins. The produced protein was assembled into Dane particles and was immunogenic in mice. The expression vector was found to be stable for at least 100 generations under non-selective conditions.