Testicular secretion of estradiol is necessary for normal spermatogenesis and male reproductive physiology in humans and rodents. The role of estradiol in nonmammalian vertebrates remains unknown, but elevated circulating estradiol has been reported in male lizards, alligators, and various bird species. We have been unable to detect circulating estradiol in male alligators; therefore, we reexamined the question of testicular production of estradiol in alligators using more rigorous assay procedures. A large pool of plasma from a male alligator was extracted and run through an HPLC column. Immunoreactive estradiol-like material eluted coincident with authentic estradiol. By using an ultrasensitive RIA and processing large volumes of male plasma (1000 μl), we were able to measure estradiol. Estradiol in male alligators ranged from 0.23 to 3.14 pg/ml, whereas estradiol in immature female alligators ranged from 14 to 66 pg/ml. Aromatase activity in microsomes from adult alligator ovarian tissue was 36.2 ± 1.6 pmol mg⁻¹ h⁻¹, whereas activity in testicular microsomes ranged between 0.92 and 2.38 pmol mg⁻¹ h⁻¹. Ovarian aromatase activity was inhibited in a concentration-dependent fashion by Fadrozole, but the essentially background activity of testicular aromatase was not inhibited at any concentration of Fadrozole. Likewise, a comparison of alligator testicular and ovarian aromatase mRNA expression gave a similar result: the ovarian expression was 600-fold higher and brain tissue was 10-fold higher than that of the testis. Circulating estradiol in male alligators is probably of extragonadal origin, and the testis produces little if any of this steroid.