This study examines the hypothesis that low serum levels of vitamin D metabolites are associated with an increased risk for colon cancer. From August through November 1974, 20,305 residents of Washington County, Maryland, donated blood for storage at -70 degrees C in a serum bank. Colon cancer was subsequently diagnosed among 57 of these residents during the period 1984-1991. Controls had donated blood in 1974 and remained free of colon cancer through the date of diagnosis of the case. Two controls were matched to each case on age (+/- 1 year), race, sex, and date of blood draw (+/- 1 month). Mean 25-hydroxyvitamin D levels were 23.6 ng/ml and 23.2 ng/ml, and mean 1,25-dihydroxyvitamin D levels were 34.7 pg/ml and 34.6 pg/ml, in cases and controls, respectively. Analysis by quintile of serum level similarly found that none of the 95% confidence intervals of the odds ratios excluded unity, and a dose-response effect was not observed. Our data provide no strong support for the hypothesis that vitamin D metabolite levels affect the subsequent risk for colon cancer.