The PLC/PRF/5 human hepatoma cells were grown in a serum-free, hormone-free, chemically defined medium; we recently characterized cell growth and hepatitis B surface antigen (HBsAg) production by these cells under these culture conditions, in the absence of interfering substances. Because of the biochemical and the antigenic properties of PLC/PRF/5 cell line showing a marked similarity to in vivo hepatocellular carcinoma, this cell line provides an in vitro system for the study both of hepatitis B virus (HBV) infection and the relationship between this infection and hepatocarcinogenesis. In this paper we describe the effect of several hormones, growth factors and drugs on growth and HBsAg production of PLC/PRF/5 human hepatoma cells. The results obtained indicate that a serum-free growth of these cells may be useful and facilitate studies either on hormone binding or on the effect of growth factors and drugs.