This in vitro study was conducted in order to determine the effects of hydrocarbons and growth phase on the phospholipid ester-linked fatty acid composition of two marine sedimentary hydrocarbon-degrading bacteria. These two strains, namely Corynebacterium sp. and Sphingomonas sp. 2MPII, were cultivated on either a simple soluble substrate (ammonium acetate) or a hydrocarbon (respectively n-eicosane and phenanthrene). The incubations were stopped at different times corresponding to point of lag (2 days), exponential (7 days) and stationary phases (21 and 56 days). The effects of growth phase and hydrophobic substrates were successfully demonstrated by a simple index, given as the sum of saturated fatty acids divided by the sum of unsaturated fatty acids ( summation operatorSFA/ summation operatorMUFA), ranging from 1.4 to 3, 0.3 to 0.6, and 0.5 to 1.0 for Corynebacterium sp., Sphingomonas sp. 2MPII, and mixed cultures, respectively. This result was validated by a principal component analysis. In pure cultures, the phospholipid fatty acid (PLFA) composition was strongly influenced by both the carbon source and the growth phase. Nevertheless, the two strains showed different "behaviors". For 2MPII, the main PLFA composition changes were observed at 2 days while they were progressive as a function of time for Corynebacterium sp. These differences could explain the evolution of PLFAs of mixed cultures.