Rhodococcus opacus 1CP, a potent degrader of (chloro-) aromatic compounds was found to utilise C10-C16 n-alkanes as sole carbon sources. Highest conversion rates were observed with n-tetradecane and n-hexadecane, whereas the utilisation of n-dodecane and n-decane was considerably slower. Thin-layer chromatography of organic extracts of n-alkane-grown 1CP cultures indicated the growth-associated formation of a glycolipid which was characterised as a trehalose dimycolate by 1H-NMR spectroscopy and mass spectrometry. Total chain lengths between 48 and 54 carbons classify the fatty acid residues as nocardiomycolic acids. The presence of two double bonds in each mycolic acid is another feature that distinguishes the corresponding trehalose dinocardiomycolates from trehalose dicorynomycolates reported for Rhodococcus erythropolis DSM43215 and Rhodococcus ruber IEGM231. R. opacus 1CP was not found, even under nitrogen limitation, to produce anionic trehalose tetraesters which have previously been reported for R. erythropolis DSM43215.