A radioimmunoassay, immunohistochemical techniques, and high pressure liquid chromatography (HPLC) methods have been developed for the study of the pentapeptide proctolin in the lobster Homarus americanus. Proctolin-like immunoreactivity is present in nearly every portion of the lobster nervous system; immunoreactivity is found in the brain, in each of the ganglia and connectives of the ventral nerve cord, and in many of the nerve roots that emerge from the cord. The greatest amounts are found in the pericardial organs, which are well known neurosecretory structures, and these structures have been selected for more detailed study. The immunoreactive material in the pericardial organs appears to be authentic proctolin. This material co-migrates with synthetic proctolin in two HPLC systems. Furthermore, a peptide that is purified from pericardial organs by HPLC is indistinguishable from synthetic proctolin in high resolution fast atom bombardment mass spectrometry. Cytochemistry reveals that the surface of the pericardial organs is densely covered with immunoreactive varicosities. No cell bodies that stain for proctolin are found in the pericardial organs, and the cells that give rise to the varicosities have not yet been located. The nerve endings in pericardial organs are capable of releasing proctolin-like material when depolarized in the presence of Ca++. These findings suggest that proctolin is a neurohormone in the lobster.