The biosynthesis of tetrodotoxin (TTX), a potent neurotoxin consisting of a 2,4-dioxaadamantane skeleton and a guanidine moiety, is an unsolved problem in natural product chemistry. Recently, the first C5-C10 directly bonded TTX analogue, 4,9-anhydro-10-hemiketal-5-deoxyTTX, was obtained from toxic newts and its carbon skeleton suggested a possible monoterpene origin. On the basis of this hypothesis, screening of predicted biosynthetic intermediates of TTX was performed using two MS-guided methods. Herein, five novel cyclic guanidine compounds from toxic newts are reported which commonly contain a cis-fused bicyclic structure including a six-membered cyclic guanidine. These structures could be biosynthetically derived from geranyl guanidine through oxidation, cyclization, and/or isomerization steps. LC-MS analysis confirmed the widespread distribution of the five novel compounds in toxic newt species. These results support the hypothesis that TTX is derived from a monoterpene.Tetrodotoxin (TTX; 1),[1] a potent and selective blocker of voltage-gated sodium ion channels, [2] causes fatal pufferfish poisoning. A recent study also advised caution with regards to low concentrations of TTX contamination in bivalve mollusks from some areas of Europe.[3] This molecule is still attracting the interest of pharmacologists and synthetic chemists.[4] TTX is distributed among various marine (such as pufferfish and snails) [5] and terrestrial (newts, frogs, and toads) [6] animals. In spite of the high interest in this unique toxin, its biosynthetic pathway is still unknown. Although toxic marine animals are considered to accumulate TTX produced by several species of bacteria, [7] bacterial gene clusters corresponding to TTX biosynthesis have not been elucidated thus far. In several studies, problems involving the isolation and cultivation of TTX-producing bacteria have been reported.[8] The debate on the internal or external origin of TTX in toxic terrestrial animals still continues, [9] but recently, no significant production of TTX or of its analogues have been observed in captivereared newts. [10] Feeding experiments performed in toxic newts by Shimizu and Kobayashi [11] resulted in no incorporation of potential TTX precursors. Although biosynthetic pathways of some natural toxins that contain guanidine group(s) have been elucidated, [12] the actual biosynthesis of TTX is still a matter of speculation.In an attempt to obtain clues for the biosynthesis of TTX, we [13] and Kotaki and Shimizu [14] identified several TTX analogues and predicted arginine and a C5 unit (isoprene or sugar) as precursors. However, we recently discovered the first C5-C10 directly bonded TTX analogue, named 4,9-anhydro-10-hemiketal-5-deoxyTTX (2), which is widely distributed among toxic newt species.[15] Based on its chemical structure (2), which consists of a guanidine and a C10 unit, we hypothesized that a monoterpene (geranyl pyrophosphate) is a precursor of TTX in terrestrial animals (Figure 1). [15] On the basis of this proposed pat...