The participation of C/EBP alpha and C/EBP beta in the transcriptional regulation of the haptoglobin (Hp) gene throughout liver development and the acute-phase (AP) response was examined. Western immunoblot analysis revealed that the relative concentrations of C/EBP alpha and C/EBP beta increased during differentiation in two nuclear protein fractions - the nuclear extract and nuclear matrix. The AP reaction was accompanied by a decrease of the relative concentration of C/EBP alpha and an increase of C/EBP beta during development in both protein fractions. Using Western analysis after DNA-affinity chromatography it was observed that a 45 kDa C/EBP alpha isoform displayed a binding affinity towards the Hp gene hormone responsive element (HRE) in both pre- and postnatal livers. In the course of the AP response DNA binding of the 45 kDa isoform was detected only in the adult, when its binding affinity decreased. The 35 kDa C/EBP beta isoform exhibited a binding affinity towards the Hp HRE after the second week from birth, whereas the AP response promoted an enhanced binding of 35 kDa isoform after the first postnatal week. These results indicate that Hp gene transcription is regulated by C/EBP alpha during normal liver development, whereas C/EBP beta is involved in the AP regulation during the later phase of differentiation and in the adult.