Ovarian and adrenal function were studied in premenopausal breast cancer patients before and at intervals during adjuvant therapy with cyclophosphamide, methotrexate, and 5-fluorouracil (CMF), CMF plus prednisone (CMFP), or CMFP plus tamoxifen (CMFPT). Amenorrhea developed within 10 months of starting therapy in 13 of 15 patients given CMF, 8 of 10 receiving CMFP, and all of 13 CMFPT-treated patients. The amenorrheic patients receiving CMF showed a reduction in their plasma total estrogens and an increase in plasma luteinizing and follicle-stimulating hormones, indicating that these cytotoxic drugs directly suppressed ovarian function. Plasma androstenedione levels, which are derived equally from the ovaries and adrenals before the menopause, were also reduced. Plasma dehydroepiandrosterone sulfate, a steroid predominantly of adrenal origin, was unaffected. CMFP-induced amenorrhea was associated with similar changes in the plasma estrogens and gonadotropins, but patients receiving this combination showed significantly greater reductions in plasma androstenedione and also decreased levels of plasma dehydroepiandrosterone sulfate. Suppression of androstenedione secretion from both the ovaries and adrenals did not affect the total plasma estrogen concentrations. Initially, CMFPT-treated patients showed significant elevations in plasma total estrogens, without a change in the gonadotropin levels. Although the plasma sex hormone-binding capacity was increased during CMFPT therapy, there was only a small reduction in the percentage of free plasma estradiol, with the result that the level of circulating unbound estrogen was increased. The plasma estrogens declined, with a corresponding increase in gonadotropins, after the onset of CMFPT-inducted amenorrhea.