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The effects of agents which are known to be differentiation inducers on a human hepatoma cell line PLC/PRF/5 were investigated. Dexamethasone (DEX), sodium butyrate (SB) or dimethylsulfoxide (DMSO) were examined. They all reduced cell proliferation but differ from each other in effect on the secretion of alphafetoprotein (AFP) and hepatitis B surface antigen (HBsAg), changes in morphology and RNA transcription. SB changed the cell from polygonal into a fibroblast-like type and decreased AFP secretion. DMSO decreased the cell size and changed AFP secretion in the same manner as SB. DEX changed the cell into a larger size, as well as increased AFP secretion. HBsAg secretion and also HB virus DNA transcription was enhanced by 3 agents. AFP and myc gene transcriptions were reduced by SB but DMSO reduced only AFP. Albumin gene transcription was enhanced by SB and DEX. These results indicate that the decrease of PLC/PRF/5 proliferation is induced through different mechanisms by these 3 agents.