Rats were injected through the carotid artery (cephalad direction) with 18.9 mu Ci of either 125I-Arg 101-Tyr 126 atrial natriuretic factor alone or together with an excess of unlabeled hormone. At 2 min after injection, all rats were fixed in vivo by perfusion and serial sections of the whole brain were processed for light microscope radioautography. The radioautographic reaction produced by 125I-atrial natriuretic factor alone was localized in all circumventricular organs (except the subcommissural organ), the smooth muscle cells and endothelial cells of arteries, arterioles, veins, venules, the endothelial cells of intraparenchymal capillaries and the epithelial cells of the choroid plexus. In rats which received 125I-atrial natriuretic factor plus an excess of unlabeled hormone, the radioautographic reaction was reduced by 70-90%. Binding sites are thus localized in regions of the brain, some of them involved in the central monitoring of blood pressure and osmolarity. In addition, the presence of binding sites in the cerebral vasculature and in the epithelium of the choroid plexus suggests that circulating ANF may play a role in the control of cerebral blood flow and in the production of vertebrospinal fluid.