The ontogeny of vitamin D3 25-hydroxylase activity has been determined in liver microsomes of rat fetuses and neonates. Production of 25-hydroxyvitamin D3 was low (0.11 pmol/g liver/h) 3 days prior to birth. Production rates were 1.2, 2.2, 1.8, and 2.8 pmol/g liver/h on Day 0, Day 2, Day 7, and Day 15, respectively. 25-Hydroxyvitamin D3 production in neonates increased sixfold from Day 15 to Day 22 to a value twice that of the mothers (17.6 pmol/g liver/h compared with 7.3 pmol/g liver/h). Activity in the maternal microsomes was constant (0.22 to 0.30 pmol/mg protein/h) except for the day of parturition (0.54 pmol/mg protein/h) and Day 22 postpartum (0.44 pmol/mg protein/h). A cytosolic factor, present as early as 3 days prior to birth, was required for vitamin D3 25-hydroxylase activity in the fetuses and stimulated the 25-hydroxylase reaction (up to 2.5-fold) in neonates and mothers. The ability of cytosol to prevent degradation of vitamin D3 was also present in the fetal stage. These data suggest that microsomal vitamin D3 25-hydroxylase activity in rat liver microsomes develops slowly and reaches full activity near the weaning stage. Since the cytosolic factor(s) is/are present in the fetal stage, the limiting component in the maturation of vitamin D3 25-hydroxylase activity in liver microsomes is the development of the cytochrome P-450 vitamin D3 25-hydroxylase.