Previous research indicates that circulating vitamin D levels are low in many otherwise healthy adults and that there is considerable seasonal variation in 25-hydroxyvitamin D [25(OH)D] concentrations at high latitudes. We examined seasonal variation in 25(OH)D levels in a sample of young adults of diverse ancestry living in the Greater Toronto Area. Three hundred and fifty-one (351) healthy young adults completed both a fall and winter visit during this study. The study was conducted over 2 y (y 1: fall 2007 to winter 2008 and y 2: fall 2008 to winter 2009). At both visits, each participant's serum 25(OH)D concentration was measured. Information was also obtained on skin pigmentation (measured via reflectometer), vitamin D intake, and extent of sun exposure. Overall, the serum 25(OH)D concentration was 54.4 ± 1.3 nmol/L in the fall and 38.4 ± 1.1 nmol/L in the winter. Concentrations differed among ancestral groups at both visits (P < 0.001), with South Asians and East Asians having substantially lower concentrations than Europeans. Skin pigmentation (r(2) = 0.14; P < 0.001), supplemental vitamin D intake (r(2) = 0.09; P < 0.001), sun exposure (r(2) = 0.04; P < 0.001), and study year (r(2) = 0.02; P = 0.017) were predictors of fall 25(OH)D concentrations. During the wintertime, serum 25(OH)D concentrations were associated with concentrations taken in the fall (r(2) = 0.45; P < 0.001), supplemental (r(2) = 0.15; P < 0.001) and dietary vitamin D intake (r(2) = 0.06; P < 0.001), and with study year (r(2) = 0.02; P = 0.009). Our study confirms that serum 25(OH)D concentrations undergo strong seasonal variation at high latitudes and are influenced by vitamin D intake, skin pigmentation, and sun exposure.