CONTEXT: The contribution of reproductive hormones to mood has been the focus of considerable research. Results from clinical and epidemiological studies have been inconsistent. It remains unclear whether alterations in serum hormone levels across the menopausal transition are linked to depressive symptoms. OBJECTIVES: To evaluate the relationship between serum hormone levels and high depressive symptoms and whether hormone levels or their change might explain the association of menopausal status with depressive symptoms previously reported in a national sample of midlife women. DESIGN: A longitudinal, community-based, multisite study of menopause. Data were collected at baseline and annually from December 1995 to January 2008 on a range of factors. Early follicular phase serum samples were assayed for levels of estradiol, follicle-stimulating hormone, testosterone, and dehydroepiandrosterone sulfate. SETTING: Seven communities nationwide. PARTICIPANTS: A community-based sample of 3302 multiethnic women, aged 42 to 52 years, still menstruating and not using exogenous reproductive hormones. Main Outcome Measure Depressive symptoms assessed with the Center for Epidemiological Studies Depression Scale (CES-D). The primary outcome was a CES-D score of 16 or higher. RESULTS: In multivariable random-effects logistic regression models, log-transformed testosterone level was significantly positively associated with higher odds of a CES-D score of 16 or higher (odds ratio = 1.15; 95% confidence interval, 1.01-1.31) across 8 years, and a larger increase in log-transformed testosterone from baseline to each annual visit was significantly associated with increased odds of a CES-D score of 16 or higher (odds ratio = 1.23; 95% confidence interval, 1.04-1.45). Less education, being Hispanic, and vasomotor symptoms, stressful life events, and low social support at each visit were each independently associated with a CES-D score of 16 or higher. No other hormones were associated with a CES-D score of 16 or higher. Being perimenopausal or postmenopausal compared with being premenopausal remained significantly associated with a CES-D score of 16 or higher in all analyses. CONCLUSIONS: Higher testosterone levels may contribute to higher depressive symptoms during the menopausal transition. This association is independent of menopausal status, which remains an independent predictor of higher depressive symptoms.