Aims: The aim of this study was to evaluate biosurfactant production by a novel marine Rhodococcus sp., strain PML026 and characterize the chemical nature and properties of the biosurfactant. Methods and Results: A novel marine bacterium (Rhodococcus species; strain PML026) was shown to produce biosurfactant in the presence of hydrophobic substrate (sunflower oil). Biosurfactant production (identified as a trehalolipid) was monitored in whole-batch cultures (oil layer and aqueous phase), aqueous phase (no oil layer) and filtered (0.2 mu m) aqueous phase (no oil or cells; extracellular) and was shown to be closely associated with growth/biomass production. Extracellular trehalolipid levels increased postonset of stationary growth phase. Purified trehalolipid was able to reduce the surface tension of water to 29mN m(-1) at Critical Micellar Concentration (CMC) of c. 250mgl(-1) and produced emulsions that were stable to a wide range of conditions (pH 2-10, temperatures of 20-100 degrees C and NaCl concentrations of 5-25% w/v). Separate chemical analyses of the intact trehalolipid and its constituents demonstrated the compound was in fact a mixture of homologues (>1180MW) consisting of a trehalose moiety esterified to a series of straight chain and hydroxylated fatty acids. Conclusions: The trehalolipid biosurfactant produced by the novel marine strain Rhodococcus sp. PML026 was characterized and exhibited high surfactant activity under a wide range of conditions. Significance and Impact of Study: Strain PML026 of Rhodococcus sp. is a potential candidate for bioremediation or biosurfactant production for various applications.