Conventional fractionation procedures used for separation and purification of pituitary FSH and LH in diverse tetrapods were employed to study pituitary gonadotropin (Gn) from five genera of snakes representing three families: Elaphe and Ptyas—Colubridae; Naja and Bungarus—Elapidae; Crotalus—Viperidae. The hormones purified from each of these snakes were shown to be relatively potent in a variety of Gn bioassays and radioreceptor assays (RRAs) in snakes but there was no clear evidence for two separate types of Gn molecule. These snake materials were also active in RRAs and bioassays (testis growth, androgen production, and ovulation) in lizards, but poor dose-response characteristics and relatively low potencies were observed in most cases. An even higher degree of species specificity was evident when snake hormones were tested in nonsquamate species, including other reptiles. Most snake Gn fractions were essentially inactive in all RRAs and bioassays employing amphibians, birds, mammals, and turtles; these assays included several that typically show broad species cross-reactivity (e.g., anuran ovulation and spermiation, 32P uptake by chick testis, and in vitro androgen production in birds and turtles). Radiolabeled Gn from a snake ( Naja) bound specifically only to gonadal receptors from snakes and lizards. Biochemical analyses of the snake Gn confirmed their glycoprotein nature but failed to show clear structural homology to either FSH or LH. Chromatographically, the purified Gn from Naja naja tended to behave predominantly like an LH and its electrophoretic mobility on polyacrylamide gels also suggested an LH-like molecule, but several problems complicated interpretation of these results. Amino acid composition of this snake Gn revealed similarities to both FSH and LH, but it was not consistently like either. Radioimmunological studies (RIAs) with several heterologous gonadotropin antisera also failed to show a consistent relatedness between snake hormones and either FSH or LH; in fact, the snake Gns are exceptional among tetrapods in showing a lack of cross-reactivity in several FSH and LH-RIA systems. Antisera were raised against Gn from two species of snake. Antiserum to Naja Gn blocked the biological and binding activities of other species of snake Gn and it also selectively neutralized the activity of FSH (but not LH) from a turtle, alligator, and bird. However, in homologous RIA, the Naja Gn showed a slight cross-reaction only with another elapid species. In a heterologous RIA (using antiserum to Ptyas Gn and 125I-labeled Naja Gn), relatively high cross-reactivity was seen with Gn from several elapids and colubrids but not viperids or any non-snake species. Thus, the snake hormones show partial relatedness to one another but are generally distinct from FSH or LH; the only evidence of cross-reaction with heterologous (non-serpentine) gonadotropin suggests that they share common immunochemical determinants with reptilian FSH. Overall, the snake gonadotropins appear to be unique among tetrapod gonadotropins in terms of their biological cross-reactivity, biochemical composition, and immunochemical properties. Data suggest that snakes may only have a single gonadotropin that does not show a clear homology to either FSH or LH.