Serum banks from large, decades-old epidemiological studies provide a valuable opportunity to explore the contributions of in utero vitamin D exposure to fetal origins of adult diseases. We compared 25-hydroxyvitamin D (25(OH)D) by race and season (two powerful predictors of vitamin D status) in sera frozen for >or= 40 years with sera frozen for <or= 2 years to determine whether 25(OH)D is stable enough to test vitamin D-related hypotheses. Data and sera came from seventy-nine pregnant women at 29-32 weeks' gestation in the Boston Collaborative Perinatal Project (CPP; 1959-66) and 124 women at 20-36 weeks' gestation in a 2003-2006 Pittsburgh cohort study. Multivariable linear regression models were used to test main and joint effects of race and season after confounder adjustment. In both cohorts, serum 25(OH)D levels were lower among black than white women (CPP 33.3 v. 46.7 nmol/l, P<0.01; Pittsburgh 47.1 v. 89.6 nmol/l; P<0.0001) and in winter than summer (CPP 32.7 v. 47.6 nmol/l, P<0.0001; Pittsburgh 66.7 v. 89.8 nmol/l, P<0.001), with no evidence of a race x season interaction in either cohort. Differences remained significant after confounder adjustment. When CPP and Pittsburgh results were compared, there was no significant difference in the race or season effects. The similarity in the relative change in 25(OH)D in these cohorts by two powerful predictors of vitamin D status suggests that, even if 25(OH)D deteriorated somewhat, it did so similarly across samples. Therefore, trends could be obtained from the decades-old serum data that would be relevant in exploring vitamin D-related hypotheses in future studies.