Concentrations of estradiol (E2) are high in the urine of male marmosets, and links between E2 and paternal behavior have been proposed in black tufted-ear marmosets, Callithrix kuhlii. However, it is not clear whether urinary E2 in male marmosets: 1) represents production of E2 associated with testicular activity, 2) is associated with adrenal steroid production, or 3) merely reflects peripheral conversion of T to E2 prior to excretion. We tested the hypothesis that urinary E2 in male marmosets represents estrogen production-associated activity in the hypothalamus-pituitary-gonad (HPG) axis. We treated adult male marmosets with gonadotropin-releasing hormone (GnRH), and used saline-treated males as controls. We collected blood and urine samples from males before and after treatment, and assayed them for testosterone (T), estradiol (E2), and cortisol (CORT). Treatment with GnRH increased circulating T and E2, and prevented decreases in levels of urinary T and E2. Moreover, changes in plasma and urinary E2 after treatment were positively correlated with post-treatment changes in T. Thus, our data are consistent with both plasma and urinary E2 in male marmosets increasing as a result of testicular stimulation. However, treatment with GnRH did not affect plasma or urinary CORT concentrations of males, suggesting that the E2 excreted by males is not of adrenal origin. We also compared urinary T, E2, and CORT levels between intact and castrated male common marmosets (Callithrix jacchus). Urinary concentrations of T and E2, but not CORT, were significantly lower in castrated than in intact males, further suggesting that E2 in male marmosets varies with testicular activity. Am. J. Primatol. 56:27–42, 2002. © 2002 Wiley-Liss, Inc.