The human hepatoma cell line, PLC/PRF/5, was shown to produce hepatitis B surface antigen (HBsAg). Immunologically reactive material was present in the supernatant tissue culture medium in significant amounts, and was associated with spherical particles approximately 20 nm in diameter. The rate of antigen production by the cells was estimated at 500 ng/day/10(6) cells by reference to a purified HBsAg standard. All immunological activity was neutralized by specific antibody and the subtype was ad. The studies reported here broaden the scope of investigations on both the in vitro production of HBsAg and the association between this antigen and primary liver cancer.