<p>A potent voltage-gated sodium ion channels blocker, tetrodotoxin (TTX; 1), has been found in various marine animals, newts and frogs. The biosynthesis of this unique toxin is still an attractive mystery. For the study of the biosynthetic and metabolic pathways of TTX, we have determined the structures of TTX analogues obtained from the toxic animals.³⁾ Recently, 4,9-anhydro-10-hemiketal-5-deoxyTTX (2) consisting of the C5–C10 directly bonded ten carbons (except guanidine) was obtained from toxic newts, thus we hypothesized a monoterpene as biosynthetic precursor of TTX.⁴⁾</p><p> In this study, on the basis of the proposed biosynthetic pathway, screening of the intermediates of TTX in early biosynthesis stage was performed using HR-LC-MS⁵⁾. Some candidate compounds, Cep-210 (3), Cep-212 (4) and Cep-242 (5) were detected in the toxic newts. These compounds were purified by several steps of column chromatography, and 3, 4 and 5 (mixture of Cep-242 (5a) and Cep-242-hemiketal (5b)) were identified as cis-fused bicyclic compounds consisting of a six-membered cyclic guanidine by NMR experiments. Based on the structures, compound 3−5 were suggested to be biosynthetic intermediates or shunts of TTX, which support our hypothetic pathway.</p><p> The study of the origin of TTX in newts, that is still controversial (internal or external), was also conducted. TTX was not detected in 70-week-old newts which were captive-reared with non-toxic food from eggs,⁶⁾ whereas all the wild-caught juvenile newts contained TTX. This result strongly suggests the external origin of TTX in newts. Further investigation of the ability of newts to accumulate TTX via oral administration was performed.</p>