Large quantities of the low-molecular-weight natriuretic material (F4), which appears after the salts when fractionated on G-25 Sephadex column, were obtained from the urine of normal man on a normal diet. The natriuretic substance in F4 was (1) untrafiltrable through a membrane with a claimed molecular-weight cut-off of 500 daltons (Amicon UMO5); (2) soluble in more polar organic solvents; (3) totally soluble in 95% acetone when specific activity was doubled; (4) relatively resistant to heating at 100 degrees C for 1 hour at a pH of 10, and to heating at 110 degrees C in 6 N hydrochloric acid for up to 90 hours under anaerobic conditions, and treatment with nitrous acid; it was less resistant to these procedures when extracted into 95% acetone; (5) not destroyed by trypsin, chymotrypsin, pronase, pepsin, leucine aminopeptidase, and subtilysin, nor was it destroyed by pepsin, leucine aminopeptidase, subtilysin, carboxypeptidase A and B, and aminopeptidase M, or by monoamine oxidase, aryl sulphatase, and beta-glucuronidase when extracted into 95% acetone. The natriuretic substance in the 95% acetone-soluble F4 was totally destroyed by incubation with prolidase. The least amount of 95% acetone-soluble F4 required to produce a significant natriuresis in the bioassay rat was that derived from a 7-min sample of urine. The maximal response was obtained from a 30-min sample of urine. Continuous i.v. infusion of the 95% acetone-soluble F4 for 40 min produced a sustained natriuresis, whereas a greater amount injected as a bolus produced an effect which was not sustained beyond 20 min.