The nuclear transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is expressed at high levels in the liver and inhibits growth in cultured cells. We have tested the correlation between C/EBPalpha levels, cell cycle proteins and hepatocyte proliferation in old and young animals as an in vivo model system in which the proliferative response to partial hepatectomy (PH) has been shown to be reduced and delayed in old animals. Here we present evidence that the expression of C/EBPalpha in old rats (24 months) differs from its expression in young animals (6-10 months) during liver regeneration. Induction of proliferating cell nuclear antigen (PCNA), a marker of DNA synthesis, occurs at 24 h after PH in young rats but is delayed and reduced in old animals. Induction of the mitotic-specific protein, cdc2 p34, is 3-4-fold less in regenerating liver of old rats than in the liver of young animals, confirming the reduced proliferative response in old animals. In young rats, the normal regenerative response involves a reduction of 3-4-fold in the levels of C/EBPalpha protein at 3-24 h. In old animals, C/EBPalpha is not reduced within 24 h after PH, but a decrease of C/EBPalpha protein levels can be detected at 72 h after PH. Induction of C/EBPbeta, another member of the C/EBP family, is delayed in old animals. Changes in the expression of C/EBP proteins are accompanied by alteration of the CDK inhibitor, p21, which is also decreased in young rats after PH, but in old animals remains unchanged. High levels of p21 protein in older animals correlate with the lack of cdk2 activation. We suggest that the failure to reduce the amount of C/EBPalpha and p21 is a critical event in the dysregulation of hepatocyte proliferation in old animals following PH.