Hydrolysis, extraction, and radioimmunoassay techniques for the estimation of excreted testosterone metabolites in the urine of saddle-back tamarins have been validated and are described. The steroids measured with the testosterone antiserum used are mostly present as glucuronides and sulfates. Immunoreactivity in high-performance liquid chromatography (HPLC) fractions of urinary extracts and in a standard mixture of cortisol, testosterone, and dihydrotestosterone (DHT) were compared. The fractions with the same retention data as testosterone accounted for the major part of the immunoreactivity . Several other immunoreactive compounds of unknown identity were present in low concentrations. These results suggest that testosterone conjugates are the major steroid metabolites measured with this method in Saguinus fuscicollis. Urinary testosterone levels of castrated males were much lower than those of intact males. Testosterone treatment of castrated males resulted in a temporary superphysiological increase in the levels of urinary testosterone and in an individually variable increase in the levels of the minor immunoreactive compounds. These results suggest that estimation of testosterone metabolite levels in urine is a valid method for the assessment of testicular activity in Saguinus fuscicollis.