About 30% of the phosphatidylglycerol in oleic acid-enriched Acholeplasma laidlawii membranes are not hydrolyzed at temperatures below 10°C by phospholipase AS from porcine pancreas. Removal of 53% of the membrane proteins by proteolysis did not reduce the size of this inaccessible phosphatidylglycerol pool. However, modification of the membrane proteins with 2,4,6trinitrobenzenesulfonic acid or glutaraldehyde did make an additional 70% of this protected pool of phosphatidylglycerol accessible to phospholipase A,. Complete hydrolysis of phosphatidylglycerol at low incubation temperatures was achieved only after heat treatment of the membranes which resulted in an extensive aggregation of intrinsic membrane proteins as visualized by freeze-etch electron microscopy. Phospholipase AZ from bee venom was more effective in hydrolyzing phosphatidylglycerol at low temperature than the pancreatic enzyme. These results show that the inaccessibility of phosphatidylglycerol is not due to resealing of isolated membranes, the presence of a crystalline phase in the membrane lipids, or a shielding effect of surface proteins. The protection against hydrolysis may be due to an interaction of phosphatidylglycerol with intrinsic membrane proteins which is stabilized at low temperatures.Increasing the temperature favors the exchange of protein-bound phosphatidylglycerol with other membrane lipids resulting in complete hydrolysis.