We have quantitated the phase preferences of all of the quantitatively significant lipids of fatty acid-homogeneous Acholeplasma laidlawii membranes by determining the effect of small amounts of each lipid on the lamellar/reversed hexagonal phase transition temperature of a phosphatidylethanolamine matrix of identical fatty acid composition using differential scanning calorimetry. We find that the incorporation of small amounts of these lipids produce effects ranging from a moderate depression to a marked elevation of the lamellar/reversed hexagonal phase transition temperature of the corresponding phosphatidylethanolamine. Thus, although the total membrane lipids from this organism form only lamellar phases under physiological conditions, the individual membrane lipids appear to exhibit a wide range of phase preferences. Phosphatidylglycerol and diglucosyldiacylglycerol seem to have relatively strong and weak preferences for the lamellar liquid-crystalline phase, respectively, while monoglucosyldiacylglycerol, and especially acyl polyprenyl glucoside, strongly prefers the reversed hexagonal phase. Most notable in this regard is the phase preference of glycerylphosphoryldiglucosyldiacylglycerol, which strongly destabilizes the reversed hexagonal phase and which we show in the accompanying paper [Lewis, R. N. A. H., & McElhaney, R. N. (1995) Biochemistry 34, 13818-13824] actually prefers the normal micellar phase in isolation. The presence of normal, lamellar, and reversed phase-preferring lipids in a single membrane has important implications for understanding the physical basis of lipid organization and biosynthetic regulation in this and possibly in other organisms. We also show that the characteristic effect of the individual A. laidlawii membrane lipids on the lamellar/reversed hexagonal phase transition temperature of the phosphatidylethanolamine matrix is not well correlated with their polar headgroup intrinsic volumes.(ABSTRACT TRUNCATED AT 250 WORDS)