This study was undertaken to investigate the utility of vitamin D3-palmitate as a nutritional supplement and thus define the intestinal absorption profile of vitamin D2 and vitamin D3 liberated after its cleavage from vitamin D3-palmitate in the human infant at various postnatal ages. The subjects for study consisted of 48 normal infants that were simultaneously administered 0.07 and 0.08 micromol/kg BW vitamin D as vitamin D3-palmitate and nonesterified vitamin D2 respectively, by orogastric tube. Blood samples were obtained before and 6, 12, and 24 h postadministration and analyzed simultaneously for vitamins D2 and D3. For data analysis, the infants were divided into two groups based on postnatal age: group 1, 1 day of age; and group 2, more than 10 days of age. Data were analyzed using the integrated peak area under the absorption curve for each subject. All subjects demonstrated the ability to absorb vitamin D after oral administration, although postnatal age as well as vitamin form had a profound effect on the absorption of vitamin D2 and vitamin D3 liberated from vitamin D3-palmitate. Nonesterified vitamin D2 is well absorbed both in very young and older infants, although absorption efficiency increases with age, perhaps due to increased bile acid secretion. Liberation of vitamin D3 from vitamin D3-palmitate was shown to increase, perhaps due to gastrointestinal tract maturation, beyond 10 days of age, probably coinciding with the secretion of intestinal esterases. Our data indicate that both forms of the orally administered vitamin approach equivalency in their abilities to elevate circulating vitamin D levels in the human infant at a postnatal age of approximately 89 days. Thus, vitamin D3-palmitate would appear not to be dietarily equivalent to free vitamin D as a nutritional source of vitamin D in the human neonate.