Large epidemiological samples, including the National Collaborative Perinatal Project (NCPP), in which blood/serum was collected during pregnancy and offspring followed longitudinally, offer the unique opportunity to examine neuroendocrine mechanisms underlying prenatal "programming" of adult health and disease. However, in order to conduct longitudinal analyses, it is critical to determine the validity of maternal prenatal samples stored over long periods. We investigated the validity of cortisol, testosterone, and their binding globulins (corticosteroid-binding globulin (CBG) and sex hormone-binding globulin (SHBG)) in maternal prenatal serum from the NCPP after over 40 years of storage. Study 1 included 64 maternal serum samples collected on the day of delivery; study 2 involved 1099 third trimester serum samples collected between gestational weeks 31 and 36. Across both studies, cortisol and testosterone concentrations were consistent with values from published studies of fresh samples collected at similar points in gestation. CBG and SHBG were present, but showed some differences from published studies. Results support the validity of cortisol and testosterone values following 40+ years of storage. Results also provide validation for future longitudinal tests of prenatal "programming" hypotheses within the NCPP. Stability of steroid hormones over decades suggests that stored samples from other longitudinal studies may also allow opportunities to investigate links between prenatal steroids and long-term offspring outcomes.