Liposomes containing either 32P-labeled diphosphatidylglycerol (cardiolipin) or 32P-labeled phosphatidyl-rac-(1)-glycerol were injected into the circulation of rats. Analysis of the liver lipids 2-3 after injection showed incorporation of the 32P label from both lipids to a lipid which had chromatographic properties identical with bis(monoacylglycero)phosphate. Stereochemical analysis of this lipid indicated that its backbone was sn-glycero-1-phospho-1'-glycerol. Cultured hamster fibroblasts (BHK cells) were incubated in a medium containing lyso[32P]phosphatidyl-rac-(1)-glycerol and the formation of radioactive lipids in the cells was followed. Bis(monoacylglycero) phosphate was the major 32P-labelled lipid formed: as much as 36.4% of the lyso[32P]phosphatidyl-rac-(1)-glycerol absorbed to the cells was converted to bis(monoacylglycero)phosphate. Similar results were obtained with lyso[32P]phosphatidyl-sn-1-glycerol as a precursor. Stereoanalysis of the bis(monoacylglycero)-[32P]-phosphate formed from either precursor indicated that this lipid was a derivative of sn-glycero-1-phospho-1'-glycerol. These results establish phosphatidylglycerol, diphosphatidylglycerol and lysophosphatidylglycerol as precursors of bis-(monoacyl-sn-glycero-1)phosphate in vivo. The mechanism of the conversion of lysophosphatidylglycerol to bis-(monoacyl-sn-glycero-1)phosphate was studied by using 32P,3H-labeled lysophosphatidyl-rac-(1)-glycerol as a precursor. Both labels were incorporated to bis(monoacylglycero)phosphate with similar efficiency, which suggests that rearrangement, rather than replacement, of the (originally acylated) sn-glycero-3-phospho moiety of the precursor is the essential reaction in the biosynthesis of the sn-glycero-1-phospho-1'-glycerol backbone of bis(monoacylglycero) phosphate.