Tetrodotoxin (TTX, 1) is a potent voltage-gated sodium channel blocker detected in certain marine and terrestrial organisms. We report here a new TTX analogue, 9-epiTTX (2), and a TTX-related compound, Tb-242B (4), isolated from the pufferfish Takifugu flavipterus and Dichotomyctere ocellatus, respectively. NMR analysis suggested that 2 exists as a mixture of hemilactal and 10,8-lactone forms, whereas other reported TTX analogues are commonly present as an equilibrium mixture of hemilactal and 10,7-lactone forms. Compound 2 and TTX were confirmed not to convert to each other by incubation under neutral and acidic conditions at 37 °C for 24 h. Compound 4 was identified as the 9-epimer of Tb-242A (3), previously reported as a possible biosynthetic precursor of TTX. Compound 4 was partially converted to 3 by incubation in a neutral buffer at 37 °C for 7 days, whereas 3 was not converted to 4 under this condition. Compound 2 was detected in several TTX-containing marine animals and a newt. Mice injected with 600 ng of 2 by intraperitoneal injection did not show any adverse symptoms, suggesting that the C-9 configuration in TTX is critical for its biological activity. Based on the structures, 2 and 4 were predicted to be shunt products for TTX biosynthesis.