It has been known for many years that mammalian pineal gland extracts contain proteinaceous substances which affect the reproductive system. The chemical mediator of antigonadotropic activity was previously hypothesized to be the non-mammalian nonapeptide arginine vasotocin. More recent evidence strongly mitigates against the presence of arginine vasotocin in mammalian pineal glands, leaving open questions about the chemical nature of the antigonadotropic peptide. We completed a large scale purification of bovine pineal neurohypophysical hormone-like peptides in which arginine vasotocin was not identified. During the course of these study a decapeptide with antigonadotropic activity was purified, subsequently isolated by serial semipreparative high performance liquid chromatography and its primary structure elucidated. Both the natural antigonadotropic decapeptide and a synthetic analog prepared by solid state methods were observed to reduce circulating levels of prolactin and luteinizing hormone. Microinfusion of the decapeptide into the lateral ventricle in rats was followed by reductions in circulating prolactin and pulsatile luteinizing hormone release. The accumulated results suggest that the confusion about the chemical nature of the putative antigonadotropic peptide of pineal gland origin may relate to the failure of most methods employed previously to separate closely associated, yet chemically and biologically dissimilar compounds as was shown to be the case with the novel antigonaodtropic decapeptide.