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Serum 25-hydroxyvitamin D (25-OHD), calcium, phosphorus, magnesium, and alkaline phosphatase levels of breast-fed infants and their mothers were studied by following 100 healthy term mother-infant pairs with different supplementation protocols of vitamin D. A pilot study in winter revealed that six of eight breast-fed infants without vitamin D supplementation had serum 25-OHD levels below the risk limit for rickets (5 ng/ml) at the age of 8 weeks. In the main study in winter groups, it was found that the 25-OHD levels were low (5.6 ± 3.7 ng/ml) at the age of 8 weeks in the unsupplemented breast-fed infants, whose mothers were given vitamin D supplementation of 1,000 IU/day during lactation (group I), compared with the levels of those infants receiving either 400 (18.0 ± 8.4 ng/ml, group II) or 1,000 IU (22.8 ± 11.2 ng/ml, group III) vitamin D (group I vs. group II or III, p < 0.001; group II vs. group III, NS). In group I 10 of 18 infants had serum 25-OHD levels <5 ng/ml compared with none of the infants in groups II and III. Yet the infants with 25-OHD levels <5 ng/ml showed no signs of clinical or biochemical rickets at the age of 8 or 20 weeks. In summer at delivery the maternal 25-OHD levels were good, but decreased thereafter. Also in summer groups, the infantile 25-OHD concentrations decreased; however, because the levels at delivery were high, they stayed in the normal range. It can be concluded that in a country with scant sunlight in winter, breast feeding alone or with maternal supplementation of 1,000 IU/day vitamin D may lead to very low 25-OHD levels in the child. A vitamin D supplement of 400 IU/day appears adequate for breast-fed infants during scarce exposure to sunlight.