The demonstration of vasotocin in the mammalian pineal gland, subcommissural organ and fetal pituitary gland by bioassay has led to hypotheses regarding the function of this hormone in various reproductive processes. Preliminary examinations of the pineal gland and subcommissural organ with a specific radioimmunoassay failed to show vasotocin immunoreactivity. The presence of vasotocin, vasopressin and oxytocin in the pineal gland, subcommissural organ and fetal neurohypophysis was therefore investigated, using three specific radioimmunoassays. Frog and chicken pituitary glands were used to validate the vasotocin radioimmunoassay. Direct measurements in diluted homogenates of pituitary glands from frogs, chickens, mid-term fetal sheep and near-term fetal seals revealed the presence of vasotocin only in the frog and chicken pituitary glands, while vasopressin and oxytocin were found in the two fetal pituitary homogenates. Vasopressin and ocytocin were measured in homogenates of rat and bovine pineal glands and in preparations of the subcommissural organ of rats and rabbits after extraction with Vycor glass powder, but no specific vasotocin immunoreactivity was observed. These results indicate a discrepancy between the reported biological activity of vasotocin in the pineal gland, subcommissural organ and fetal pituitary gland and the immunoreactivity of this material, which can at present only be explained by the presence of a peptide which is structurally closely related to, but not identical with, vasotocin.