plsX (acyl-acyl carrier protein [ACP]:phosphate acyltransferase), plsY (yneS) (acyl-phosphate:glycerol-phosphate acyltransferase), and plsC (yhdO) (acyl-ACP:1-acylglycerol-phosphate acyltransferase) function in phosphatidic acid formation, the precursor to membrane phospholipids. The physiological functions of these genes was inferred from their in vitro biochemical activities, and this study investigated their roles in gram-positive phospholipid metabolism through the analysis of conditional knockout strains in the Bacillus subtilis model system. The depletion of PlsX led to the cessation of both fatty acid synthesis and phospholipid synthesis. The inactivation of PlsY also blocked phospholipid synthesis, but fatty acid formation continued due to the appearance of acylphosphate intermediates and fatty acids arising from their hydrolysis. Phospholipid synthesis ceased following PlsC depletion, but fatty acid synthesis continued at a high rate, leading to the accumulation of fatty acids arising from the dephosphorylation of 1-acylglycerol-3-P followed by the deacylation of monoacylglycerol. Analysis of glycerol 3-P acylation in B. subtilis membranes showed that PlsY was an acylphosphate-specific acyltransferase, whereas PlsC used only acyl-ACP as an acyl donor. PlsX was found in the soluble fraction of disrupted cells but was associated with the cell membrane in intact organisms. These data establish that PlsX is a key enzyme that coordinates the production of fatty acids and membrane phospholipids in B. subtilis.