Growth of Pseudomonas oleovorans GPol in continuous culture containing a bulk n-octane phase resulted in changes of the fatty acid composition of the membrane lipids. Compared to citrate-grown cells, the ratio of C18 to C16 fatty acids and the ratio of unsaturated to saturated fatty acids increased as a result of growth on octane. Trans-unsaturated fatty acids, which are rarely found in bacteria, were formed during continuous growth of P. oleovorans on octane. Moreover, the mean acyl chain length and unsaturated fatty acids also increased as the growth rates increased both in octane-grown and citrate-grown cells. Differential scanning calorimetry measurements of extracted lipids showed the transition temperature of membrane lipids from octane-grown cells increased from about 24°C to 32°C as the growth rate increased, whereas cells grown on citrate showed a constant transition temperature of about 6°C at all growth rates tested, indicating a decrease of membrane lipid fluidity in octane-grown cells. Because alkanes are known to increase bilayer fluidity by intercalating between lipid fatty acyl chains, the increased transition temperature of the lipids of cells grown on octane may be a physiological response of P. oleovorans to compensate for the direct effects of octane on its cellular membranes.