The 3-layer immunoperoxidase-bridge technique was used to study the distribution of neurophysin and vasopressin in the neurosecretory neurons of rats homozygous and heterozygous for diabetes insipidus (Brattleboro strain). In the homozygous rats there was a marked hypertrophy of the hypothalamic magnocellular structures when stained either for neurosecretory material or neurophysin-like antigens. Neurophysin was present in both the paraventricular (PVN) and supraoptic nuclei (SON) of homozygous and heterozygous animals. Less than half of the cells in the PVN and SON were stained for neurophysin. This observation was less apparent when histochemical stains were used to visualize the distribution of neurosecretory material. Although it is generally considered that the homozygous Brattleboro rat does not synthesize vasopressin, a positive reaction was observed in the PVN and SON when anti-[8-lysine]-vasopressin serum was employed in the immunohistochemical procedure.