Background and Aims: Vitamin D is important for calcium absorption and bone growth. Hypovitaminosis and vitamin D deficiency have been observed in developed and developing countries including several in the Middle East. The prevalence of vitamin D deficiency was investigated in Isfahani male and female high school students in Iran in 2004. Methods: In a cross-sectional study, 318 students (153 boys and 165 girls) aged 14–18 years were selected by multistage random sampling. Data were collected by interview and serum 25-hydroxyvitamin D (25-OHD) and parathyroid hormone (PTH) levels were measured. Vitamin D deficiency was defined as serum 25-OHD <20 and <32 ng/ml in which the mean serum PTH began to increase in our population. Results: The prevalence of vitamin D deficiency according to 25-OHD <20 ng/ml in all subjects was 46.2% (72.1% in females and 18.3% in males). Vitamin D deficiency in female students was about four times more common than in males (OR = 3.9, 95% CI = 1.88–3.91, p < 0.001). According to 25-OHD <32 ng/ml, the prevalence of vitamin D deficiency in all students was 72.2% (95.2% in females and 49% in males). Conclusions: Vitamin D deficiency is common in high school students, especially in girls. Avoidance of sunshine or inadequate dietary intake of vitamin D may be the main causes.

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