A pituitary glycopeptide whose amino acid sequence was previously identified has now been recognized as the final portion of the precursor to arginine vasopressin and its associated neurophysin. Immunocytochemical techniques with antiserums against this 39 amino acid peptide and vasopressin were used to study their distribution in the rat central nervous system. The peptide is located in vasopressin-synthesizing cells in the neurosecretory magnocellular nuclei. Positively stained fibers project from the magnocellular nuclei through the median eminence to the posterior pituitary. Studies of the homozygous Brattleboro rat, which is known to be deficient in the production of vasopressin and its related neurophysin, also show the absence of immunoreactivity to this peptide. These immunocytochemical data strongly indicate that the peptide is synthesized with vasopressin.