A study was undertaken to investigate the aerobic bacterial flora of the sea-cucumber Holothuria atra, and the bacterial population of the animal's immediate environment and food source (marine sediment). Intestinal and sediment samples were used as sources of inoculum to isolate bacteria. Dilution plating of samples indicated that numbers of culturable bacteria were greater in the hindgut than in the foregut, but in both cases less than that found in the surrounding sediment. Additionally, higher numbers of viable bacteria were obtained from the foregut of a starved animal than from that of a normal feeding animal. From the isolation study, 43 strains were selected for identification and characterisation by 16S ribosomal DNA sequence analysis and limited phenotypic testing. Of these isolates, 24 were phylogenetically related to members of the genus Vibrio and neighboring taxa. Other isolates included members of the genus Bacillus, the alpha and gamma subclasses of the Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides lineage, and the order Actinomycetales. Sequence analysis and hybridization screening of a 16S rDNA clone library from the gut content of H. atra showed that cloned sequences represented members of the alpha, beta and gamma subclasses of the Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group and the actinomycetes. No clone 16S rDNA sequence was found to be identical to the sequence of any of the isolates from this study, or that of any described species for which sequence data is available. The majority of clones for which 16S rDNA sequence information was obtained i.e. 67.5% originated from organisms belonging to the beta subclass of the Proteobacteria. A number of sequences originating from members of the family Pasteurellaceae were also demonstrated.