Mutants of Escherichia coli containing a defective sn-glycerol 3-phosphate acyltransferase are conditionally defective in the synthesis of acylglycerol phosphate (acylglycerol-P). Incubation of a deep rough derivative of one of these plsB strains with 1-[3H]oleoylglycerol-32P resulted in the binding of up to 70 nmol of oleoylglycerol-P per 100 nmol of cellular phospholipid. The binding was dependent on time, oleoylglycerol-P concentration, and the quantity of cells employed. The rate and extent of oleoylglycerol-P binding was affected by the deep rough mutation. The altered phospholipid composition due to oleoylglycerol-P binding was without consequence on cell growth and viability, but caused the appearance of intracellular multilamellar structures. Use of the double-labeled oleoylglycerol P demonstrated that the entire molecule was bound to the cell. Intact [3H]-oleoylglycerol-32P was converted to phosphatidylethanolamine and phosphotidyl-glycerol at a rate about 40% of that of de novo phospholipid synthesis. These data demonstrate the transmembrane movement of oleoylglycerol-P to the inner surface of the cytoplasmic membrane and suggest that it may become possible to supplement plsB strains of E. coli with acylglycerol-P's.