A population residing in the approximate area of Kalamazoo, MI (latitude north, 42 degrees 17' 29''; longitude west 85 degrees 35' 14''), was examined to determine the influence of seasonal variation on human 25-hydroxyvitamin D3 serum levels. Males and females, ranging in age from 16--64 yr of age and judged normal based on laboratory evaluation and physical examination, participated in the 12-month study. Measurement of 25-hydroxyvitamin D3 serum levels was made by high performance liquid chromatography. A correlation coefficient of 0.776 (P = 0.003) was obtained by comparing average monthly 25-hydroxyvitamin D3 serum levels to average monthly temperatures. A comparison of approximated monthly amounts of sunlight to average monthly 25-hydroxyvitamin D3 serum levels produced a correlation coefficient of 0.747 (p = 0.005). In addition, changes in 25-hydroxyvitamin D3 levels for the population examined fitted a model that demonstrated a relationship to sex and a highly significant periodic relationship to time.