Addition of epidermal growth factor (EGF) to Chang liver cells in a low serum culture causes a long-lasting, two- to three-fold increase in the acetylation of nuclear proteins. This EGF effect is manifested before an increase of cell proliferation in response to EGF. Studies by SDS-polyacrylamide gel electrophoresis and isoelectric focusing show that both histones and acid-insoluble proteins of lower molecular weights are acetylated upon administration of EGF. Tumor promoter teleocidin, which inhibits internalization and nuclear accumulation of EGF, acts antagonistically with EGF in enhancing the acetylation of nuclear proteins. Lysosomal inhibitor chloroquine enhances nuclear accumulation of EGF but has no significant effects on the stimulation of nuclear protein acetylation by EGF. These data appear to suggest that the EGF-enhancement of the acetylation of nuclear proteins is mediated by the steps such as an internalization or nuclear accumulation of EGF or EGF-receptor complex.