The hydrothermal-vent gastropod Alviniconcha aff. hessleri from the Kairei hydrothermal field on the Central Indian Ridge houses bacterium-like cells internally in its greatly enlarged gill. A single 16S rRNA gene sequence was obtained from the DNA extract of the gill, and phylogenetic analysis placed the source organism within a lineage of the epsilon subdivision of the Proteobacteria. Fluorescence in situ hybridization analysis with an oligonucleotide probe targeting the specific epsilonproteobacterial subgroup showed the bacterium densely colonizing the gill filaments. Carbon isotopic homogeneity among the gastropod tissue parts, regardless of the abundance of the endosymbiont cells, suggests that the carbon isotopic composition of the endosymbiont biomass is approximately the same as that of the gastropod. Compound-specific carbon isotopic analysis revealed that fatty acids from the gastropod tissues are all (13)C enriched relative to the gastropod biomass and that the monounsaturated C(16) fatty acid that originates from the endosymbiont is as (13)C enriched relative to the gastropod biomass as that of the epsilonproteobacterial cultures grown under chemoautotrophic conditions. This fractionation pattern is most likely due to chemoautotrophy based on the reductive tricarboxylic-acid (rTCA) cycle and subsequent fatty acid biosynthesis from (13)C-enriched acetyl coenzyme A. Enzymatic characterization revealed evident activity of several key enzymes of the rTCA cycle, as well as the absence of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the gill tissue. The results from anatomic, molecular phylogenetic, bulk and compound-specific carbon isotopic, and enzymatic analyses all support the inference that a novel nutritional strategy relying on chemoautotrophy in the epsilonproteobacterial endosymbiont is utilized by the hydrothermal-vent gastropod from the Indian Ocean. The discrepancies between the data of the present study and those of previous ones for Alviniconcha gastropods from the Pacific Ocean imply that at least two lineages of chemoautotrophic bacteria, phylogenetically distinct at the subdivision level, occur as the primary endosymbiont in one host animal type.