Atrial natriuretic peptide (ANP) represents a family of related peptides originally isolated from cardiac atria that have potent natriuretic, diuretic, and vasorelaxant properties. ANP has previously been localized in neurons of the rat brain in regions subserving cardiovascular functions and fluid/electrolyte balance and has been localized in astroglia of the canine brain. To determine whether ANP is present in astrocytes of the human brain and to validate the canine model for future studies, human brain tissue was obtained from autopsy cases with no brain damage or neurological or vascular disease. Human brains were obtained less than 3 h postmortem, and anterior cingulate and striate cortices were dissected following perfusion or immersion fixation. Immunohistochemical processing utilized antibodies against the processed form of ANP (ANP IV, ANP104-128) and against rat proANP (amino terminus) and the avidin-biotin-peroxidase technique. Isolated, strongly ANP-immunoreactive protoplasmic astrocytes were observed in all layers of the cingulate and striate cortex gray matter. ANP-positive fibrous astrocytes were observed in the white matter. Additionally, distinctive immunopositive astrocytes were found both within and immediately subjacent to the glia limitans. Antibody against the prohormone stained only protoplasmic astrocytes and sublimitans astrocytes and processes. In addition to the astroglia, ANP was detected in scattered multipolar neurons in the cerebral gray matter. These results provide additional evidence for diversity of peptide localization in astrocytes and suggest roles for ANP in the local regulation of cerebral blood flow, blood-brain barrier permeability, or cerebrospinal fluid volume.