We measured longitudinal levels of vitamin D in unsupplemented Malawian infants at 0 (birth), 2, 12, 15, 18 and 24 months of age. Matched maternal plasma and breast milk vitamin D(2) and D(3) levels were also measured at delivery and 2 months postpartum. Vitamin D was measured using isotope-dilution liquid chromatography tandem-mass spectrometry. Vitamin D(3) levels in children were 36% of adult levels at birth, 60% of adult levels at age 2 months, and at par with adult levels by 12 months of age. This adult-equivalent level is subsequently maintained through age 24 months and consisted of a 98% molar ratio of vitamin D(3). Vitamin D levels in breast milk were below the limit of detection, 0.1 ng/ml. Breast milk of unsupplemented Malawian mothers is a poor source of vitamin D.