Homogenates of kidneys from hydropenic and volume-expanded rats were subjected to gel filtration with Sephadex G-25. A fraction of the eluate coincident with the fourth UV peak was injected into the aorta of rats with one kidney excluded. A fraction eluting before the albumin peak was utilized as a control. Significant natriuresis and diuresis were observed after infusion of the fraction obtained from volume-expanded kidneys but not after infusion of the fraction from hydropenic kidneys or the control fraction. The natriuresis occurred in in the absence of changes in mean blood pressure, hematocrit, plasma sodium and potassium, glomerular filtration rate, and potassium excretion. The response was apparent immediately after infusion and persisted for up to 150 min. These results verify the existence of a low molecular weight natriuretic substance which may be preferentially bound to the kidney after its volume-stimulated release into the circulation.