Serum samples drawn from 34 women in the early part of both their first and their second pregnancies were assayed for estradiol (E2), percentage of free E2, and sex hormone-binding globulin-binding capacity (SHBG-bc). Subjects were participants in the Collaborative Perinatal Study conducted by the National Institute of Neurological and Communicative Disorders and Stroke (Bethesda, MD) to evaluate factors related to adverse pregnancy outcome. All pregnancies were full term, and no offspring had a congenital malformation. After adjustment for week of pregnancy, the percentage of free E2 was 9% higher (two-sided P = .007) and the amount of free E2 was 17% higher (two-sided P = .03) in first-pregnancy sera. Total E2 was also 7% higher in first-pregnancy sera after adjustment for week of pregnancy; SHBG-bc was 7% lower after adjustment for week of pregnancy and 9% lower after simultaneous adjustment for week of pregnancy and weight at the start of pregnancy; but these differences were not statistically significant. These findings confirm our previously published hypothesis that the early part of a woman's first pregnancy differs endocrinologically from her second and may provide further insight into pregnancy-related risk factors for testis cancer and cryptorchidism and into the protection afforded by first full-term pregnancy against breast cancer.