This paper describes the development and validation of a plasma and urinary gonadotropin immunoassay for golden lion tamarins (Leontopithecus rosalia), an endangered New World callitrichid primate. The assay is derived from a macaque chorionic gonadotropin assay and was validated for both plasma and urine samples in L. rosalia. Levels of immunoreactive LH/CG in lion tamarin urine were highly correlated (r = + 0.98) with gonadotropin bioactivity. Immunoreactive LH/CG levels were examined in two contexts: in the urine of adult females and in the plasma of adult males after administration of estrogen. Peaks of gonadotropin excretion were detected in samples collected from nonpregnant adult females. The peaks occurred immediately prior to cyclic elevations in urinary estrogen excretion. Plasma LH/CG concentration in males measured 24 and 48 hours after a single 50 μg injection of estradiol benzoate were significantly lower than levels at these time points measured after control treatment. Together, the results of this study point to the utility of the gonadotropin assay for monitoring reproductive function in both female and male lion tamarins.