Intraluminal concentrations of bile acids are low in newborn infants and increase rapidly after birth, at least partly due to increased bile acid synthesis rates. The expansion of the bile acid pool is critical since bile acids are required to stimulate bile flow and absorb lipids, a major component of newborn diets. The purpose of the current studies was to determine the mechanism responsible for the increase in bile acid synthesis rates and the subsequent enlargement of bile acid pool sizes (BAPS) during the neonatal period, and how changes in circulating hormone levels might affect BAPS. In the hamster, pool size was low just after birth and increased modestly until 10.5 days post-partum (dpp). BAPS increased more significantly (3-fold) between 10.5 and 15.5 dpp. An increase in mRNA and protein levels of cholesterol 7-hydroxylase (Cyp7a1), the rate limiting step in classical bile acid synthesis, immediately preceded an increase in BAPS. In contrast, levels of oxysterol 7-hydroxylase (Cyp7b1), a key enzyme in bile acid synthesis by the alternative pathway, were relatively elevated by 1.5 dpp. FXR and SHP mRNA levels remained relatively constant at a time when Cyp7a1 levels increased. Finally, though simultaneous increases in circulating cortisol and Cyp7a1 levels occurred, precocious expression of Cyp7a1 could not be induced in neonatal hamsters with dexamethasone. Thus, the significant increase in Cyp7a1 levels in neonatal hamsters is due to an FXR/SHP- and cortisol-independent increase in Cyp7a1 levels.