We have studied gonadal function in five patients (three men and two women) who received high-dose methotrexate (HD-MTX) alone and in 12 patients (seven men and five women) who received HD-MTX and vincristine (VCR) as postoperative adjuvant therapy for osteosarcoma. Testicular function was assessed by determination of testicular size, sperm concentration, and serum follicle-stimulation hormone (FSH), luteinizing hormone (LH), and testosterone levels while evaluation of ovarian function was based on menstrual history and serum FSH, LH, estradiol, and progesterone levels. The two women who received HD-MTX and all five women who received HD-MTX and VCR had regular cyclic menses not only after therapy but also during treatment. Serum FSH, LH, estradiol, and progesterone levels were normal in all women studied. Among the ten men studied, testicular function was normal in the six patients evaluated initially several months after the completion of therapy. In contrast, severe oligospermia was noted among four men evaluated during and immediately after treatment. From serum samples obtained before, during, and after treatment we have determined that approximately one half of the men who received HD-MTX alone or in combination with VCR develop transient testicular failure associated with a significant increase in serum FSH but not LH levels. Sperm concentration and serum FSH levels then return to normal after completion of chemotherapy.