The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and between individual S. velum organisms suggested that one species of symbiont is dominant within and specific for this host species. Phylogenetic analysis of the 16S sequences of the symbionts indicates that they lie within the chemoautotrophic cluster of the gamma subdivision of the eubacterial group Proteobacteria.Procaryote-eucaryote associations in which marine invertebrates harbor chemoautotrophic bacteria as endosymbionts appear to be widespread in marine habitats such as deep-sea hydrothermal vents and coastal sediments (8,15). In such symbioses, the procaryotes utilize the energy released by the oxidation of reduced inorganic substrates, such as hydrogen sulfide, to fix carbon dioxide via the CalvinBenson cycle (7, 13). The hosts appear to derive nutrition from their endosymbionts and in turn provide the symbionts simultaneous access to the substrates from anoxic and oxic environments which are necessary for energy generation. Maintenance of such intracellular symbionts presents a novel metazoan acquisition of procaryotic energy generation and autotrophic carbon fixation.While the existence of chemoautotroph-invertebrate symbioses is now generally accepted, little is actually known about the symbionts observed in the tissues of any of the hosts because none have been cultured. Comparison of rRNA sequences has greatly facilitated the identification of bacteria, including unculturable microorganisms, and the elucidation of their natural relationships (38). Phylogenetic analysis of 16S rRNA sequences enabled Distel et al. (12) to establish that the chemoautotrophic symbionts of the hydrothermal vent tubeworm and five species of bivalves of the subclass Lamellibranchia are related and cluster in the gamma subdivision of the Proteobacteria (formerly purple photosynthetic bacteria), one of the 11 major groups of eubacteria (30).In this investigation we sought to establish the phylogenetic relationships and the species specificities of the symbionts of the protobranch bivalve Solemya velum Say, an Atlantic coast clam which has been studied as a shallowwater model of invertebrate-chemoautotroph associations (7, 9, 10). The phylogenetic placement of the S. velum symbionts, to date limited to sequence analysis of the 5S rRNA, indicates that these symbionts also fall in the Proteobacteria gamma subdivision (31). However, the small size of * Corresponding author. t Present address: