FSH and/or LH deficiency in the second decade of life remains difficult to diagnose with testing at a single point in time because of the partial lack of hormone as well as the dynamic and inherently variable aspects of the pubertal process. A longitudinal study of gonadotropin excretion, therefore, was carried out in 78 normal boys and 157 male patients, aged 10-28 yr, with relative or absolute deficiencies of FSH and/or LH. Seven hundred and fifty-five timed urine samples were extracted with acetone, concentrated, and subjected to RIA. The results from patient groups with multiple tropic hormone deficiencies or isolated gonadotropin deficiency were clearly different from those of normal boys and individuals with constitutional delay in puberty. However, multiple samples obtained over a 2-yr period and, in selected cases, until the late teenage years may be required to diagnose gonadotropin deficiency in some patients, even using stringent predictive modeling criteria.