Membranes from rabbit aorta and from rabbit and rat kidney cortex possess high-affinity (Kd = 10(-10) M) specific binding sites for atrial natriuretic factor (ANF). Similar high-affinity sites are present in an established cell line from pig kidney, LLC-PK1. Results of fractionation studies indicate that the receptors are localized in the plasma membrane of these tissues. The binding is time-dependent and saturable. An excellent quantitative correlation was found between the affinity of synthetic ANF and analogs of intermediate activity to aorta membranes and the half-maximal concentration needed for relaxation of rabbit aorta rings contracted by addition of serotonin. Furthermore, the binding affinity of the receptor in kidney membranes is consistent with the concentration required for in vivo natriuresis in the rat. Biologically inactive synthetic ANF fragments and other peptide hormones such as angiotensin II and vasopressin do not significantly inhibit binding. These data suggest that the receptors for ANF in vascular and renal tissues are responsible for mediating the physiological actions of this peptide in these target tissues.