Studies on a chemical extraction and purification procedures are described for rat atrial natriuretic factor. This factor—first detected by injection of crude rat atrial heart muscle homogenates—induces a powerful diuretic and natriuretic response when injected into nondiuretic assay rats. Studies on the properties of purified natriuretic factor suggest that it is of a proteinaceous nature. It is efficiently extracted by dilute acetic acid, readily degraded by protease, and recoverable after gel filtration at a fractionation range corresponding to less than 6000 daltons. Dose-response studies show that at maximal doses the response is a 10- to 15-fold increase in urine output and a 30- to 40-fold increase in urinary sodium excretion. These responses are characteristically of rapid onset (1-2 min) and decay (10-15 min).