The indirect immunofluorescence technique of Coons and collaborators was used to study the distribution of substance P-like and luliberin (luteinizing hormone-releasing hormone)-like immunoreactivity in the eminentia mediana (eminentia medialis or infundibulum) of rats, monkeys, and human beings. In rats, abundant luliberin-positive fibers were present in the external layer (mainly lateral parts) whereas almost no substance P-immunoreactive nerves were observed in this region. In contrast, in the external layer of the primate eminentia medialis a dense plexus of substance P-positive nerve terminals was observed close to the blood vessels supplying the anterior pituitary gland. Luliberin-immunoreactive fibers were also present, but with a more even distribution all over the eminentia medialis and in lower numbers. The present findings indicate that substance P may play a role in the control of hormone secretion from the anterior pituitary, either by being released into the portal vessels, i.e., acting as a releasing or inhibitory hormone, or by an action as local regulator (modulator or transmitter) at the level of the eminentia medialis. Thus, in agreement with many earlier studies, substances other than the "classical" releasing and inhibitory hormones may be important for the regulation of the pituitary gland. Furthermore, there may exist marked species differences with regard to the type of substances involved in the central control of the pituitary.