Treatment of primary cultured hepatocytes from adult (6-month-old) rats with epidermal growth factor (EGF) results in a marked elevation in DNA synthesis, a response that is markedly attenuated in cells of aged (24-month-old) animals. Recently we demonstrated that this age-related attenuation is associated with reduced activation of extracellular signal-regulated kinase (ERK) in response to EGF treatment. In order to gain further insight into the mechanisms responsible for the age-related decline in this proliferative response, we investigated the expression and/or activities of several other regulatory molecules important for G1 to S phase progression in EGF-stimulated young and aged hepatocytes. Induction of cyclin D1 and activation of cyclin-dependent kinase 2 (CDK2) by EGF were found to be diminished in the aged cells. In young cells, prior treatment with rapamycin inhibited the induction of DNA synthesis and activation of CDK2 to levels similar to those seen in aged cells without inhibiting ERK activity and cyclin D1 expression. This suggested that a distinct, ERK-independent, rapamycin-sensitive pathway might also contribute to the proliferative response in hepatocytes and be subject to age-related alterations. Further studies demonstrated that activation of p70 S6 kinase (p70S6k), a rapamycin-sensitive event, following EGF treatment was 40% lower in aged hepatocytes relative to young cells, although the kinetics of activation did not differ in the two age groups. Western blot analysis for p70S6k expression revealed similar levels of proteins in young and aged cells. From these findings, we conclude that deficiencies in both the ERK and p70S6k signaling pathways contribute to the age-related decline in the proliferative response of hepatocytes.