Plasma was obtained by an isovolaemic exchange procedure from rats undergoing sustained blood volume expansion (experimental) and from normovolaemic but otherwise similarly prepared rats (control). Four plasma fractions, showing absorption at 280 nm, were separated by G-50 Sephadex gel filtration, freeze dried and reconstituted for assay in hydropaenic rats for natriuretic activity. Only the large protein fraction (molecular weight exceeding 30 000) of experimental plasma produced a significantly greater natriuresis than the corresponding fraction from either control plasma or a fractionated 5.6% albumin solution. The respective averaged peak differences in sodium excretion were 4 and 3.5 μequiv./min per gram kidney weight. This additional natriuretic activity was attributed to a humoral factor, either a protein or a protein-bound moiety, generated by vascular expansion.