The production and specific productivity of hepatitis B surface antigen (HBsAg) in recombinant Chinese, hamster ovary (CHO) cells were increased by 81% and threefold, respectively, when supplemented with 1.5% dimethyl sulfoxide (DMSO) in the culture medium. To investigate the mechanism of DMSO effect on HBsAg production improvement, HBsAg mRNA level was measured. by real-time PCR. HBsAg mRNA was increased by about 1.5-fold at 1.5% DMSO. The increase could derive from the increase of HBsAg gene copy number, the improvement of HBsAg mRNA stability, or the acceleration of HBsAg gene transcription: It was found that HBsAg gene copy number was not significantly changed in the cells stimulated with DMSO. HBsAg mRNA stability of cells with 1 DMSO treatment was also not obviously different from control, and the mRNA half-life of 5.58 h in the cells at 1.5% DMSO was comparable to that of 5.36 h in the control culture. DMSO resulted in 80% increase in HBsAg gene transcription activity assessed using a nuclear run-on transcription assay. It could be deduced that the acceleration of HBsAg gene transcription is the main cause of HBsAg production improvement.