Some physicochemical properties of partially purified hypothalamic material from the spontaneously hypertensive rat, and of plasma from man and the rat, have been characterized using a validated cytochemical bioassay which measures the ability of biological fluids to stimulate fresh guinea-pig kidney glucose-6-phosphate dehydrogenase (G6PD) after 2 min of exposure to the test substance, as an indication of their ability to inhibit Na+/K+ adenosine triphosphatase (Na+/K+-ATPase) after 4-6 min of exposure. The G6PD-stimulating activity of both hypothalamic extract and plasma is soluble in water and insoluble in chloroform. During electrophoresis the activity from both sites appears in the same fractions and travels considerably further than lysine. After high-pressure liquid chromatography the activity of hypothalamic extract appears in a discreet fraction which does not absorb u.v. light. The activity of both the hypothalamic extract and plasma survives boiling and acid hydrolysis, but is substantially inhibited by prior incubation with digoxin antibody. From ultrafiltration studies, the substance responsible for the ability to stimulate G6PD appears to have a molecular weight of less than 500. The G6PD-stimulating activity of hypothalamic extracts was destroyed by ashing and by base hydrolysis. The ability of plasma of high activity to stimulate G6PD is considerably increased by incubating at 37 degrees C for 15 min and destroyed by incubation for 45 min. It is concluded that these and several other previously noted similarities suggest that the cytochemically assayable Na+/K+-ATPase-inhibiting/G6PD-stimulating activity in the plasma and hypothalamus may be due to the same ouabain-like substance.