Reuber H-35 hepatoma cells were examined for their ability to synthesize protein in vitro, especially to produce alpha-fetoprotein (AFP). The presence of AFP in the culture supernatant solution was determined immunologically by the micro-Ouchterlony method. Charge heterogeneity of AFP was examined electrophoretically in continuous gradient polyacrylamide microgels. With regard to the duration of culture, there was no remarkable change in the ratio of two peaks of AFP, and which came out as a major combined peak and a similar peak by PAS staining on the condition of added SDS. These findings indicated that Reuber H-35 hepatoma cells had potential to produce two charge variants of AFP in vitro.