Deuterium nuclear magnetic resonance (NMR) techniques were employed to study the effect of sterols on the composition and dynamics of the membrane lipids of Mycoplasma capricolum, a natural fatty acid auxotroph that requires sterols for growth. The membrane lipids of cells grown in modified Edwards medium supplemented with cholesterol, oleic acid (OA), and palmitic acid (PA) were composed primarily of phosphatidylglycerol (PG) (60%) and cardiolipin (CL) (35%). The incorporation of cholesterol and the cellular OA/PA ratio increased nonlinearly with increases in exogenous cholesterol level, whereas the levels of phospholipid increased only slightly. At the growth temperature, 37 degrees C, the residual deuterium quadrupole splittings were found to be 43-46 kHz for cells grown with (7,7,8,8-2H4) PA and 1.25 micrograms/ml (30 mol%) to 10 micrograms/ml (50 mol%) cholesterol, respectively, similar to that found in the cholesterol/lecithin binary dispersions of similar cholesterol contents. Deuterium T2e of these samples were found to be 170 +/- 10 microseconds and were independent of cellular cholesterol content. In comparison, T2e of the corresponding lipid extracts were longer (320-420 microseconds) and dependent on cholesterol content. Thus, lipid-protein interactions in the cell membrane is the dominant mechanism responsible for the reduced T2e. At lower temperatures, spectra indicative of the coexistence of gel and liquid-crystalline states were observed for cells having low cholesterol levels. For both cell membrane and membrane lipid extract containing 50 mol% cholesterol, T2e was found to be constant at the temperature range from 15 to 40 degrees C. On the other hand, T2e of cell membrane containing 30 mol% cholesterol decreased linearly at 3.2 microseconds/degrees C. T2e of the corresponding lipid extract showed much stronger temperature variation. Cells containing 39 mol% lanosterol were found to have a quadrupole splitting of 39 kHz, broader than that of the cholesterol-free lecithin dispersion (less than 30 kHz) but less than that of cell membrane containing 30 mol% cholesterol (43 kHz). T2e of the lanosterol sample was found to be 130 +/- 10 microseconds which decreased linearly at a slope similar to that observed for the low cholesterol sample. Therefore, although lanosterol appeared to be capable of modulating cell membrane physical properties it is less effective than cholesterol. When growth rates were correlated with NMR parameters, we found that the membranes of faster growing cells were also more ordered. In contrast, the T2e of the cells of M. capricolum seemed to be maintained at a relatively constant value around 170 microseconds.