We examined epidermal growth factor (EGF)- and epinephrine-stimulated mitogen-activated protein kinase kinase (MEK) 1 and MEK2 activities, DNA polymerase  activity, and EGF-stimulated E2F DNA binding activity in primary cultured hepatocytes from 6- and 24-mo-old rats. MEK stimulation by either EGF or epinephrine was not altered with aging. However, stimulation of DNA polymerase  activity by these agents was 70% and 50% lower, respectively, in cells of aged compared with cells of young rats, consistent with a lesser increase in [³H]thymidine incorporation. EGF-stimulated E2F (a transcription factor that regulates expression of the DNA polymerase  gene) binding to DNA was reduced with age. PD-098059, a specific inhibitor of MEK, inhibited EGF-stimulated MEK1 and MEK2 activities in hepatocytes from 6- and 24-mo-old rats. Although PD-098059 inhibited EGF-stimulated DNA synthesis in hepatocytes from 6-mo-old rats, it had no effect in 24-mo-old rats. Thus the age-related impairment appears to occur before E2F activation, and signal transduction sequences other than the mitogen-activated protein kinase pathway may be involved in stimulated DNA synthesis in hepatocytes from old rats.