When adult rat hepatocytes were cultured in plastic Petri dishes in a medium containing insulin and glucagon, supplementation with epidermal growth factor (EGF) had a pronounced effect on their viability, morphology, and biochemical integrity. Transmission and scanning electron microscopic studies showed that after 1 week cells denied EGF accumulated numerous non-electron-dense bodies and filamentous whorls, had irregular nuclei, and exhibited atypical cell surfaces. In contrast, cells grown for 2-3 weeks in the presence of EGF had well-preserved cellular organelles and remained as an epithelial-like monolayer. After 3 weeks EGF-exposed cultures were still inducible for liver-specific tyrosine aminotransferase, and both rat albumin and rat transferrin were recoverable from the culture medium. Virtually no viable cells were present at 3 weeks in EGF-deprived cultures.