Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic Acid (LBPA), belongs to the group of polyglycerophospholipids and is a structural isomer of phosphatidylglycerol (PG). It is enriched in intraluminal vesicles (ILVs) of late endosomes (LEs) and lysosomes attaining approximately 15% of total phospholipids. In LEs, BMP promotes the formation of ILVs due to its cone-shaped structure. Located in the membrane of ILVs, the negatively charged BMP attracts positively charged proteins such as acid hydrolases to the surface of these vesicles. Thereby, it promotes the degradation and sorting of various lipids such as (glyco)sphingolipids, ceramides, and cholesterylester (CE). BMP itself is highly resistant to acid hydrolases due to its atypical sn1:sn1 stereoconfiguration of the glycerol backbone. Despite its biological importance, little is known about the biogenesis of BMP. Currently, hypothetical pathways propose that the formation of BMP starts either from acylation of PG or phospholipase D (PLD) - mediated transphosphatidylation reactions. Here, we investigated whether PLD can affects the biogenesis of BMP. This enzyme degrades phosphatidylcholine to phosphatidic acid (PA) and choline. In the presence of primary alcohols, a second reaction may occur attaching the alcohol to the phosphate residue of PA. Accordingly, PLD may generate bis(diacylglycerol)phosphate (BDP) in the presence of PC and diacylglycerol as PA acceptor. BDP may act as precursor of BMP. Using specific PLD inhibitors, we demonstrate that inactivation of this enzyme strongly affects cellular BMP levels. Furthermore, lipidomic analysis revealed that PLD inhibition causes accumulation of lysophosphatidylcholine (LPC), marking LPC as putative donor of lysophosphatidic acid generating BMP in the presence of monoacylglycerol as acceptor alcohol.In conclusion, our data suggest that PLD in involved in BMP synthesis and might therefore directly affect ILV biogenesis, cargo sorting and degradation.