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For the first time, alcohol extracts of Atelopus hoogmoedi from the Guiana Shield in Suriname and Guyana were analyzed for the presence of tetrodotoxin (TTX) and of its analogues by high resolution hydrophilic interaction liquid chromatography/mass spectrometry. One specimen from Suriname was found to contain TTX and 4-epiTTX. Using a monoclonal antibody-based immunohistochemical staining technique, TTX was localized mainly in the granular glands and epithelium of the skin, but not in internal organs except liver showing weak TTX-positive reaction. In two specimens collected in Guyana, none of the toxins were detected.Toads of the genus Atelopus are distributed in Central and South-America, many species are listed as critically endangered.They are known to contain tetrodotoxin (TTX) and various analogues of the toxin.For the first time, specimens of Atelopus hoogmoedi from the Guiana Shield in Suriname and Guyana have been investigated for the presence of TTX.By immunohistochemical technique, TTX has been detected in the granular glands and epithelial cells of the skin.