Human FSH [follicle stimulating hormone] iodinated with the aid of lactoperoxidase (125I-hFSH) was used to study gonadotropin binding to a variety of reptilian and avian gonadal tissues; gonads were examined from 5 species of turtle (Chrysemys, Pseudemys, Chelydra, Trionyx and Chelonia), a snake (Thamnophis), a lizard (Anolis) and 2 birds (chicken and duck). All gonadal tissues, including testes and ovaries and isolated corpora lutea from a turtle, showed high affinity, saturable, specific binding of the 125I-h FSH. The binding was dependent on tissue concentration, time and temperature; full inhibition curves were obtained with concentrations of unlabeled hFSH ranging from about 6-1200 ng/ml (.apprxeq. 10-10 to 10-7 M). Maximal binding of 125I-hFSH varied from 5-30% of the total labeled ligand; it was highest in lizard and snake testes and lowest in bird testes (these values are comparable to those obtained with mammalian gonadal tissues). Nongonadal tissues.sbd.thyroid, liver and spleen.sbd.from snakes and turtles did not show specific binding. Radioligand binding analyses with a variety of purified pituitary hormones from 4 mammalian species revealed that competition for binding of 125I-hFSH by reptilian gonadal tissues was associated with a high degree of hormonal specificity. High concentrations of growth hormone, prolactin and thyrotropin had no appreciable effect; FSH of ovine, bovine, rat and human origin all inhibited the binding of 125I-hFSH at relatively low concentrations. These preparations of FSH ranged in potency from 0.2-3 times that of a highly purified hFSH reference preparation. The binding of 125I-hFSH to turtle and avian tissues and porcine granulosa cells was also highly specific for FSH; the potency of LH from the 4 mammalian sources was usually <0.1% of the homologous FSH. All preparations of LH showed appreciable activity in inhibiting the binding of 125I-hFSH to snake and lizard tissues. Some preparations of LH, especially bovine, were almost as potent as the homologous FSH in inhibiting 125I-hFSH binding to snake testes. Mammalian FSH and LH may act at the same binding sites in some reptilian tissues, and the hormonal specificity of reptilian binding sites varies among species.