Background/Aims: Recent evidence suggests that CCAAT/enhancer binding protein (C/EBP) transcription factors may regulate hepatocyte terminal differentiation. Methods: To explore this possibility, the present study looked for variations in the expression or DNA binding activity of different C/EBP isoforms during rat postnatal liver development and determined which of the C/EBPs were expressed by adult hepatocytes in primary culture. Results: In intact rats, hepatocyte proliferation is active for 2–3 weeks after birth. During this period of postnatal liver growth, several liver-specific functions emerge and C/EBPα, β, and δ isoforms are induced. Nuclear expression of the 36-kilodalton C/EBPδ protein increases immediately after birth, followed first by increases in the 38-kilodalton C/EBPβ protein expression and then by increases in the 42-kilodalton C/EBPα protein expression. Changes in C/EBP DNA binding activity accompany developmental increases in C/EBP proteins. Messenger RNAs of all three C/EBP isoforms are expressed by mature hepatocytes in primary culture. Conclusions: Specific C/EBP isoforms are induced differentially during the course of rat postnatal liver development. Young adult rats and cultured adult hepatocytes express all three C/EBP isoforms. These results are consistent with (but do not prove) the theory that variations in C/EBP expression and function help regulate hepatocyte terminal differentiation.