To elucidate the sulphur sources for chemoautotrophy by the symbiotic bacteria of a vestimentiferan tubeworm, Lamellibrachia satsuma, living in Kagoshima Bay at depths of 80–100 m, we analysed the sulphur isotopic ratios of the animal tissues and compared them with environmental sulphur species collected in the field. Animals that had been maintained in an aquarium for over a year and supplied a known sulphur source were also investigated. The gas emitted from volcanic source in Kagoshima Bay contained rather heavy sulphide (+12·7 to +22·9‰ δ34S) compared with deep-sea hydrothermal vent systems (0 to +5‰). The tissue of the tubeworms contained very light sulphide (−21·5 to −25·9‰). It is inferred from the analysis of the aquarium-maintained specimens that the fractionation by the tubeworm or its symbiont was <1·5‰. The sulphur source assimilated by the tubeworms in the field is therefore inferred to have δ ratio −19·1 to −24·6‰. This means that only 9·7 to 25·0% of the sulphur in the worm tissues can be derived from the volcanic gas and the rest must come from other sources, such as microbial activity in the bottom sediment.