Using the surface antigen gene of the hepatitis B virus, and the promoter and terminator sequences of the yeast pho5 gene as a model system, a series of closely related expression plasmids were constructed to investigate the effect of vector type, genetic background of host strains and the presence of transcription terminator on the expression of heterologous gene in yeast. Plasmids carrying the replication origin of the 2 mu plasmids were found to be much more stable than those either independently or simultaneously carrying ars1 sequences. Gene expression was also higher with 2 micron-based plasmids. Yeast selection marker (trp1 or leu2) and therefore the host strains used did not have significant effects on gene expression. Addition of transcription terminator sequences downstream to the HBsAg gene also contributed only limited increases in gene expression levels.