Tetrodotoxin (TTX), one of the best known marine toxins with a unique chemical structure and specific action of blocking sodium channels of excitable membranes, has an obscure biogenesis. Previous studies suggested its exogenous origin in pufferfish and implicated a red calcareous alga (Jania sp.) as a potential toxin transmitter. To clarify the true origin of TTX, we investigated the toxin production of Pseudomonas sp., a bacterium previously isolated from Jania sp. The bacterium was cultured in a medium of 3% NaCl and 1% polypeptone at 25°C for 12 days without aeration. Toxins were extracted from the culture broth via acidification, boiling, evaporation, methanol extraction, and charcoal column chromatography. Using fluorometric HPLC, thin-layer chromatography (TLC), mouse bioassay, alkaline degradation, and mass spectrometry (EI and FAB), two compounds (A, 200 μg; B, 55 μg) were isolated. HPLC and TLC showed A and B were indistinguishable from reference TTX and anhydrotetrodotoxin (anhydTTX). Mouse bioassay confirmed A caused typical TTX intoxication (770 ng/mouse, median death time matching TTX) and B had potency close to anhydTTX. Alkaline degradation yielded a compound identical to authentic 2-amino-6-hydroxymethyl-8-hydroxyquinazoline (C8 base), a degradation product of TTX and its derivatives. FAB mass spectra of A and B exhibited ions at m/z 320 and 302, assignable to [M+H]+ of TTX and anhydTTX, respectively. All data confirmed that A and B are identical to TTX and anhydTTX, respectively, and are genuine products of Pseudomonas sp.