OBJECTIVE: To examine the vitamin D status of 4-month-old exclusively breastfed infants supplemented with 400 IU daily of vitamin D and to determine whether there was any seasonal variation in serum 25-hydroxyvitamin D (25(OH)D) levels of infants. METHODS: In this cross-sectional study, serum calcium, phosphorus, alkaline phosphatase, parathyroid hormone, and 25(OH)D levels of 143 exclusively breastfed 4-month-old infants supplemented daily with 400 IU of vitamin D were measured in a temperate latitude, Izmir, Turkey, between May 2008 and April 2009. A questionnaire on demographic characteristics of infants and mothers, vitamin D supplementation of infants after birth, mothers' multivitamin supplementation, dressing habits, and consumption of dairy products during pregnancy was used. RESULTS: Vitamin D deficiency (≤ 50 nmol/L) and insufficiency (51-74 nmol/L) were determined in 40 (28%) and 55 (38.5%) infants, respectively. During winter days, serum 25(OH)D levels were <20 ng/mL in 45.4% of infants and <10 ng/mL in 10.6% of infants. Season of blood sampling, compliance of vitamin D supplementation, maternal education level, and consumption of dairy products were highly predictive of serum 25(OH)D levels in multiple linear regression analysis (P < .05). The use of the Pearson correlation test found a statistically significant negative correlation between 25(OH)D and parathyroid hormone levels (r = -0.419, P < .001). CONCLUSIONS: Despite supplementation with 400 IU of vitamin D daily, the rate of vitamin D deficiency was worryingly high in 4-month-old exclusively breastfed infants living in Izmir, Turkey. So, additional studies are needed to clarify optimal amount of vitamin D supplementation to the infants, especially during winter days.