Simultaneous coexistence of differentiated, proliferating and redifferentiated hepatocytes occurs during normal liver regeneration (LR). The aim of the present work was to study the time course of the capacity of the liver to form bile during synchronized LR. Following two-thirds partial hepatectomy (PH) in rats, i.v. administration of the ribonucleotide reductase reversible inhibitor hydroxyurea (HU) was used to transiently block liver cells at G1/S boundary. Experiments were performed at 0 and 4 hours, and 1, 3 or 7 days after releasing HU-induced inhibition. Bile acid pool size was determined by collecting bile samples over 24 hours. Initial (first hour) bile flow and bile acid output were increased early on during synchronized LR as compared with the values found in non-hepatectomized control animals. These values were thereafter (1 day) reduced, but increased again at 3 days after halting HU infusion. The time course of bile acid depletion and changes in bile flow were very similar in control and synchronized LR, except that in the latter a more important early reduction in bile flow and bile acid output was found. Shortly after PH, part of the bile acid pool was lost, but this was quickly restored, soon (1 day) reaching a net bile acid pool size very similar to that found in control rats. The highest pool size relative to liver weight was found on day 1, when bile acid output and bile flow reached their lowest values. Additional experiments were performed using in situ perfused regenerating rat livers in which stepwise infusion of taurocholate (TC) was carried out. PH alone modified neither the bile acid-independent (BAIF) nor the bile acid-dependent fraction of bile flow (BADF). However, in normal LR, the BAIF decreased on day 1 and recovered at 7 days, while in synchronized LR it remained depressed up to 7 days. The BADF was only reduced during the early phase of normal LR and did not change significantly in synchronized LR. The maximal secretion rate (SRmax) for TC, as expressed per gram of remaining liver tissue, was not affected immediately after PH, but a marked reduction was observed on day 1 in both normal and synchronized LR. Afterwards, SRmax was quickly restored in both synchronized LR and, although in a slower way, normal LR. These results suggest that synchronization of LR involves changes in the time required to the recovery of specific liver functions such as bile formation.