Since Acholeplasma laidlawii can be restricted to incorporating fatty acids from the growth medium into its membrane lipids, it is possible to study the effects of the length of the acyl chains on the properties of the membrane of the organism. A. laidlawii strain A-EF22 was grown with mixtures of one perdeuterated saturated fatty acid and one monounsaturated fatty acid. The average length (<Cn>) of the acyl chains in the membrane lipids varied from 14.6 to 19.9, and the degree of unsaturation ranged from 21 to 79 mol %. 2H nuclear magnetic resonance (NMR) spectra were recorded on whole cells, on intact membranes, and on lipids extracted from these membranes. It was found that the NMR spectra for all three cases were very similar, yielding deuterium quadrupolar splittings typical for the lamellar liquid-crystalline phase (L alpha) found in model membrane systems. The use of a perdeuterated acyl chain as a reporter molecule allowed for the calculation of order parameters averaged over the entire system. These measurements yielded a wide range of average order parameters varying from 0.136 to 0.186 for the membranes and from 0.137 to 0.181 for the extracted lipids. From the order parameters the average acyl chain length can be calculated, which is related to the average membrane thickness. This value ranged from 23.2 to 30.6 A. When either the order or the membrane thickness of the intact membranes was compared to that of the extracted lipids, only slight or even undetectable differences were found. This implies that the proteins associated with the membranes do not have any large effect on the overall packing of the membrane lipids, even though the membrane thickness varied by approximately 8 A over the series studied. A decrease in the ordering of the acyl chains was observed when the length of the acyl chains incorporated from the growth medium was increased in either the membranes or the extracted lipids. This decrease correlated with the decrease in the fraction of monoglucosyldiacylglycerol (MGlcDAG) found in the membrane. Since both the average order and the membrane thickness varied, it is proposed that by changing the mole fraction of MGlcDAG the organism regulates either the membrane curvature energy or the permeability, both of which are related to lipid packing in the bilayer.