SUMMARY Acetone-dried rat posterior pituitary glands were extracted with 0·1mhydrochloric acid and the soluble proteins fractionated by Sephadex gel exclusion and ion exchange chromatography. Three proteins had chromatographic and biochemical properties expected of neurophysins and also cross-reacted immunologically (as demonstrated by microimmuno-diffusion and -electrophoresis in agarose) with an anti-serum raised against porcine neurophysin-II. The three proteins were named neurophysin-I, -II and -III in order of their electrophoretic mobility on starch-gel and accounted for approximately 8, 70 and 22% respectively of the total neurophysin present in the gland. The assignment of the major rat posterior pituitary lobe protein as a neurophysin (neurophysin-II) is confirmed by its ability to bind oxytocin and [8-arginine]-vasopressin. A minimum molecular weight of 10056 was calculated for neurophysin-II from its amino acid analysis and based upon the presence of one molecule of methionine and one molecule of tyrosine per molecule of protein. The stoicheiometry of the neurophysins relative to the neurohypophysial hormones is discussed.