Hepatitis B virus (HBV) DNA was isolated from the recombinant plasmid pA01 -HBV and recircularized . Immediately after introduction of this DNA into mouse fibroblasts (NIH 3T3) we observed increasing release of hepatitis B surface antigen (HBsAg) into the culture medium. Later production of HBsAg declined to a lower but constant level. No dominant selective marker and foreign promoter were necessary in this system, which therefore can be used for the study of regulation of HBsAg expression.