The distribution of atrial natriuretic factor (ANF)-like reactivity was examined in rat brain and heart by immunohistochemistry. Immunostaining in heart was confined to atrial myocytes. In the hypothalamus, ANF-absorbable immunoreactivity was observed in magnocellular perikarya of the paraventricular and supraoptic nuclei, and in their projections to the neural lobe of the pituitary gland. No staining was seen in the preoptic or arcuate hypothalamic nuclei or in brain stem nuclei as previously reported by other investigators. The patterns of reactivity for ANF reported here is similar to that observed for neurophysins (NPs). Comparison of sequence data between rat ANF-28 and bovine NPs revealed three regions of 3 amino acid homology between these hypothalamic peptides. Preabsorption of the ANF antiserum with Affigel-coupled bovine NP I also resulted in complete elimination of all "ANF-immunoreactivity" in both atrium and hypothalamus. Cross-reactivity of the ANF antiserum with bovine NP I and II was further confirmed by Western blot analysis. Our findings suggest that ANF antisera can cross-react with NPs if they are directed against the shared antigenic epitopes; complete elimination of staining by preabsorption of the antibody with the immunogen, therefore, does not guarantee authenticity of localization. These observations may have relevance to an earlier study which reported on the existence of ANF-immunoreactivity in oxytocin neurons of the hypothalamus.