The localization of ANF-like immunoreactivity in the ventricle of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques, using an antiserum against synthetic ANF (Arg 101-Tyr 126). At the optic level, an appreciable number of positive cardiocytes was observed in the frog ventricle. Electron microscopic studies showed that all secretory granules present in ventricular cardiocytes contain immunoreactive ANF. The immunoreactive material has been characterized by Sephadex G-50 gel chromatography and reverse phase high performance liquid chromatography (RP-HPLC). After gel filtration, ANF-like immunoreactivity eluted in 3 peaks. The major immunoreactive peak corresponded to high molecular weight material, while one peak co-eluted with synthetic ANF (Arg 101-Tyr 126). Further analysis of frog ventricular extracts by RP-HPLC revealed that the low molecular weight material has the same retention time as synthetic ANF, suggesting a high degree of sequence homology between amphibian and mammalian ANF. These results indicate that in amphibians, ventricular cardiocytes synthesize a peptide immunologically and chemically related to mammalian ANF.