Objective: The prime objective of this study is to determine the origin of Tetrodotoxin in the pufferfish Takifugu oblongus. Methods: Ultracentrifugation was performed to separate the tissues into various cellular fractions. Mouse bioassay was done to analyse the toxicity of different fractions from skin, liver and ovary of T. oblongus. The toxic fractions were subjected to HPLC-UV detection, to detect the presence of tetrodotoxin and quantify it. Results: Cytosolic fractions of skin, liver and ovary were extremely toxic and the presence of tetrodotoxin was proved on comparison with the retention time of standard toxin. Conclusions: These results suggest that the origin of tetrodotoxin could be endogenous or biosynthesized by the pufferfishes themselves. Clarifying the contradictions about the origins of TTX in puffers would allow researchers to accurately identify its role and toxicogenesis of the organisms which possess tetrodotoxin. © 2017, Association of Pharmaceutical Teachers of India. All rights reserved.