Vitamin D metabolite levels were measured in 174 normal children throughout the year. 25-hydroxyvitamin D3(25(OH)D3) and 24,25-dihydroxyvitamin D3(24,25(OH)2D3) levels showed a seasonal variation; both levels were higher in summer than in winter (p<0.001 for both). There was a fall in the 1,25-dihydroxyvitamin D (1,25(OH)2D) level in August and September (p<0.001), which coincided with a rise in mean 25(OH)D3and 24,25(OH)2D3levels. An inverse correlation was seen between 1,25(OH)2D and 24,25(OH)2D3levels (r=-0.233;p<0.01). 25(OH)D3levels increased with age only for winter values (<3 years, 11.70 ± 3.98 ng/ml; 3-11 years, 18.38 ± 1.65 ng/ml; >11 years, 23.60 ± 4.60 ng/ml) while 1,25(OH)2D and 24,25(OH)2D3levels did not show an age-related difference. Intake of multivitamins had an interesting effect on 25(OH)D3and 24,25(OH)2D3levels in the winter but not in the summer; the endogenous metabolite levels were lower in the vitamin supplemented children [25(OH)D3: 23.05 ± 7.35versus15.77 ± 5.51 ng/ml,p<0.001; 24,25(OH)2D3: 2.30 ± 1.11versus1.66 ± 0.88 ng/ml,p< 0.05]. Children studied in the winter who were not receiving supplemental vitamins were older than those who did receive the vitamins (7.26 ± 2.64versus5.42 ± 3.17 years;p<0.01). Sixteen of the children had both winter and summer measurements. Their 25(OH)D3and 24,25(OH)2D3levels showed the same seasonal variation as the overall group data, while their 1,25(OH)2D levels showed no consistent pattern. Our data suggest that establishing normal pediatric values for 25(OH)D3, 1,25OH)2D, and 24,25(OH)2D3requires consideration of season, age, and supplemental vitamin D intake, and that such information be available to investigators in order to make meaningful interpretations of vitamin D metabolite levels in disease states.