Phospholipids are key constituents of microbial membranes and account for a substantial fraction of microbial P, while P-free betaine lipids provide a pathway for membrane synthesis independent of P. The aim of this study was to test if modulation of relative amounts of phospholipids vs P-free betaine lipids is a response to P availability. To examine responses of membrane lipids to P availability, we added P or P + C to an extremely P-poor sandstone-derived soil (total P = 41 μg g−1) and a higher P shale-derived soil (total P = 201 μg g−1). By using D2O labelling and LC-MS we were able to disentangle membrane lipid synthesis dependent on P (i.e. 2H incorporation into phospholipids) from membrane lipid synthesis independent of P (i.e. 2H incorporation into P-free betaine lipids). In unamended soils, P-free betaine lipids were almost 60% of the membrane lipid pool of the P-poor sandstone-derived soil, but only 10% of the higher-P shale-derived soil. Hence, to produce a unit of membrane lipid, the microbial population of sandstone soil required only 54% as much P as the microbial population of shale soil. Four days after adding P to the sandstone soil, phospholipids accounted for a larger proportion of the pool and synthesis of membrane lipids while pool and synthesis of P-free betaine lipids decreased. The increased allocation to phospholipids in sandstone soil amended with P likely reflected amelioration of P limitation given that P addition did not affect pools or synthesis of phospholipids versus betaine lipids in the higher-P shale soil. Collectively these data support the idea that having membranes with a high proportion of P-free betaine lipids is a key adaptive trait of microbial populations of P-poor soil, while the plasticity afforded by membrane lipid remodelling enables short-term fine-tuning of membrane lipid composition to P availability.