Current practice for achieving local control of testicular relapse in males with acute lymphoblastic leukemia (ALL) includes the use of 2,400-rad testicular radiation. Although this therapy is known to cause germ cell depletion, it has been assumed that it does not alter testicular secretion of testosterone. To test this assumption, we measured gonadotropin and testosterone levels in seven boys with ALL who had been treated with radiation for clinically apparent testicular relapse. In four of seven boys, testicular relapse was bilateral with overt involvement of one testicle and microscopic involvement of the other. Three of these four boys demonstrated delayed sexual maturation, and in addition to elevated follicle-stimulating hormone (FSH) concentrations, testosterone levels were low and luteinizing hormone levels were elevated compared with controls. These data indicate that boys with overt testicular leukemia who are treated with 2,400-rad testicular radiation are at risk for Leydig cell dysfunction. However, the relative contributions of radiation, prior chemotherapy, and leukemic infiltration to this dysfunction remain to be clarified.