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Background: An epimeric form of 25-hydroxyvitamin D 3 (25(OH)D 3 ) has recently been detected in clinical samples, with relatively high levels in infants. Little is known on 3-epi-25(OH) D 3 formation and physiological function. Our objective was to study dynamics of 3-epi-25(OH)D 3 formation during infancy. Methods: 25(OH)D 3 and 3-epi-25(OH)D 3 levels were measured by liquid chromatography-tandem mass spectrometry in 22 preterm (aged 34-37 wk), 15 early preterm (aged <34 wk), and 118 term infants up to 2 y of age. All infants were prescribed vitamin D 400 IU/day after the first week of life. results: At birth, 3-epi-25(OH)D 3 levels were 3 (1-7) nmol/l, <10% of total 25(OH)D 3 . From the second week to 3 mo of age, both 25(OH)D 3 and 3-epi-25(OH)D 3 increased, with highest 3-epi-25(OH)D 3 contribution in early preterm infants (up to 55% of total 25(OH)D 3 vs. 36% in term infants, P < 0.0001). After 3 mo of age, 3-epi-25(OH)D 3 normalized to <10% in all infants. conclusions: At birth, all infants showed low contribution of 3-epi-25(OH)D 3 , increasing the week after starting vitamin D supplementation, until 3 mo of age. Highest levels of 3-epi-25(OH)D 3 were found in early preterm infants, supporting the hypothesis that hepatic immaturity plays a role in 3-epi-25(OH) D 3 formation.