We studied the effects of frozen storage on (pro)vitamin concentrations in EDTA-plasma and whole blood. Aliquots from 55 samples were analyzed before storage and after 3, 6, 12, 24, 36 and 48 months at -20 degrees C. Dramatic decreases occurred for EDTA-plasma concentrations of vitamin E between 6 and 12 months, vitamin A, total carotenoids and beta-carotene after 1 year, and whole blood niacin. A smaller decrease was observed for folic acid at 1 year of storage, but the level remained constant thereafter. The vitamins D, B6, B12 (EDTA-plasma), B1 and B2 (whole blood) showed no decline during 4 years of storage. With the exception of folic acid, the observed decreases varied considerably among subjects. Therefore using EDTA-plasma stored longer than 1 year at -20 degrees C will result in highly attenuated odds ratios when assessing the relationship between vitamin A, carotenoids, or vitamin E with a given disease. Attenuation will also occur when using niacin concentrations in whole blood stored for 4 years at -20 degrees C.