Diethylstilbestrol (DES), a synthetic estrogen widely prescribed to pregnant women in the mid-1900s, is a potent endocrine disruptor. Prenatal DES exposure has been associated with reproductive disorders in women, but little is known about its effects on endogenous hormones. We assessed the association between prenatal DES exposure and reproductive hormones among participants from the Harvard Study of Moods and Cycles (HSMC), a longitudinal study of premenopausal women aged 36–45 years from Massachusetts (1995–1999). Prenatal DES exposure was reported at baseline (43 DES exposed and 782 unexposed). Early follicular-phase concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol were measured at baseline and every 6 months during 36 months of follow-up. Inhibin B concentrations were measured through 18 months. We used multivariable logistic and repeated-measures linear regression to estimate odds ratios (OR) and percent differences in mean hormone values (β), respectively, comparing DES exposed with unexposed women, adjusted for potential confounders. DES-exposed women had lower mean concentrations of estradiol (pg/ml) (β=−15.6%, 95% confidence interval (CI): −26.5%, −3.2%) and inhibin B (pg/ml) (β=−20.3%, CI: −35.1%, −2.3%), and higher mean concentrations of FSH (IU/I) (β=12.2%, CI: −1.5%, 27.9%) and LH (IU/I) (β=10.4%, CI: −7.2%, 31.3%), than unexposed women. ORs for the association of DES with maximum FSH>10 IU/I and minimum inhibin B<45 pg/ml – indicators of low ovarian reserve – were 1.90 (CI: 0.86, 4.22) and 4.00 (CI: 0.88–18.1), respectively. Prenatal DES exposure was associated with variation in concentrations of FSH, estradiol and inhibin B among women of late reproductive age.