Aims: Our goal was to find a novel, biosurfactant-producing bacterium from Pacific Ocean deep-sea sediments. Methods and Results: An oil-degrading biosurfactant-producing bacterium TW53 was obtained from deep-sea sediment, and was identified through 16S rDNA analysis as belonging to the genus Rhodococcus. It lowered the surface tension of its culture to 34.4 mN m(-1). Thin layer chromatography (TLC) showed that the crude biosurfactants of TW53 were composed of lipopeptides and free fatty acids (FA). The lipopeptides were purified with column chromatography and then hydrolysed with 6 mol l(-1) HCl. Gas chromatography-mass spectrometry analysis showed that the hydrolyte in the hydrophobic fraction contained five kinds of FA with chain lengths of C-14-C-19, and C16H32O2 was a major component making up 59.18% of the total. However, 3-hydroxyl FA was not found, although it is usually found in lipopeptides. Silica gel TLC revealed that the hydrolyte in the hydrophilic fraction was composed of five kinds of amino acids; consistently, ESI-Q-TOF-MS analysis confirmed the composition results and provided their sequence tentatively as Ala-Ile-Asp-Met-Pro. Furthermore, the yield and CMC (critical micelle concentrations) of purified lipopeptides were examined. The purified product reduced the surface tension of water to 30.7 mN m(-1) with a CMC value of 23.7 mg l(-1). These results suggest that Rhodococcus sp. TW53 produces a novel lipopeptide that we have named rhodofactin. Conclusion: The deep-sea isolate Rhodococcus sp. TW53 was the first reported lipopeptide-producing bacterium of this genus. The lipopeptides had novel chemical compositions. Significance and Impact of the Study: Rhodococcus sp. TW53 has potential in the exploration of new biosurfactants and could be used in bioremediation of marine oil pollution.