Several fullerene-based nanomaterials generate reactive oxygen species that can damage cells. In this study, we investigated the effect of buckminsterfullerene (C60) introduced as colloidal aggregates in water (nC60) on bacterial membrane lipid composition and phase behavior. Pseudomonas putida (Gram-negative) and Bacillus subtilis (Gram-positive) responded to nC60 by altering membrane lipid composition, phase transition temperature, and membrane fluidity. P. putida decreased its levels of unsaturated fatty acids and increased the proportions of cyclopropane fatty acids in the presence of nC60, possibly to protect the bacterial membrane from oxidative stress. Fourier transform infrared spectroscopy measurement of intact bacterial cells showed slightly increased phase transition temperatures (Tm) and increased membrane fluidity for cells grown in the presence of high, growth-inhibiting concentrations (0.5 mg L(-1)) of nC60. B. subtilis responded to a low dose of nC6o (0.01 mg L(-1)) by significantly increasing the levels of iso- and anteiso-branched fatty acids (from 5.8 to 31.5% and 12.9 to 32.3% of total fatty acids, respectively) and to a high, growth-inhibiting concentration of nC60 (0.75 mg L-1) by increasing synthesis of monounsaturated fatty acids. In contrast to P. putida, B. subtilis response was a decrease in Tm and an increase in membrane fluidity. These findings represent the first demonstrated physiological adaptation response of bacteria to a manufactured nanomaterial, and they showthat response inlipid composition and membrane phase behavior depends on both the nC60 concentration and the cell wall morphology.