The small, cell-wall-less prokaryote Acholeplasma laidlawii strain A-EF22 could grow with membrane lipids having an average acyl chain length Cn varying over 14.5- almost 20 carbons by exogenous supplementation with selected fatty acids. For 16 < Cn < 18, the cells grew with lipids containing 100% (mol/100 mol) monounsaturated acyl chains, whereas for Cn < 16 and Cn > 18, cell growth only occurred with gradually lower fractions of unsaturated chains. Cn was actively increased and decreased by chain elongation or de novo fatty acid synthesis upon incorporation of short-chain and long-chain fatty acids, respectively. The membrane lipid composition was strongly affected by the acyl chain length and unsaturation, and the metabolic responses are readily explained as a regulation mechanism based on the established phase equilibria of the individual lipids in the A. laidlawii membrane. Monoglucosyldiacylglycerol (Glc-acyl2-Gro) was the dominating lipid with short chains but the fraction of this lipid decreased with increasing Cn, correlating with the decreasing lamellar to nonlamellar phase transition temperatures for this lipid. The fractions of diglucosyldiacylglycerol (Glc2-acyl2Gro) and phosphatidylglycerol (PtdGro), forming lamellar phases only, increased with increasing Cn over the entire chain-length interval. A weaker correlation was usually observed between the relative amount of a lipid and the extent of chain unsaturation; however, the fractions of Glc2-acyl2Gro and PtdGro increased clearly with an increasing degree of unsaturation. Moreover, the synthesis of the nonbilayer-forming lipids acyl2Gro and monoacyl-Glc-acyl2Gro was strongly stimulated by a high degree of chain saturation. Concomitantly, the phase equilibria of Glc-acyl2Gro are shifted towards lamellar phases at the growth temperature. The fraction of the three potentially nonbilayer-forming lipids varied over 10-80% (mol/100 mol) total lipids as a function of the acyl chain composition. The combined molar fractions of the three phospholipids increased strongly with chain unsaturation. However, the fraction of phosphate moieties in the different lipids was constant over the entire chain-length interval. It is concluded that the regulation of the membrane lipid composition aims at maintaining similar phase equilibria and surface charge densities of the lipid bilayer. The size of A. laidlawii cells was changed in a systematic manner and correlated qualitatively with the packing properties of the lipids. Cell diameters were increased by an increase in acyl chain length and saturation, and was affected by additives such an n-dodecane and acyl2Gro.