Accurate and precise determination of phospholipid composition by 31P NMR spectroscopy requires correct assignments and adequate spectral resolution. Because temperature and pH may affect chemical shifts (delta), our first aim was to establish the temperature coefficient (Deltadelta/DeltaT) of common phospholipid classes when using sodium cholate as detergent. This parameter can then be used to aid in resonance assignments. The second goal was to investigate the pH dependence of delta so that, in addition to temperature, pH control can be used to minimize spectral overlap. For phosphatidylcholine, sphingomyelin, dihydrosphingomyelin and phosphatidylglycerol, delta values were invariant with pH and temperature. Whereas the Deltadelta/DeltaT for phosphatidylinositol was 4 x 10(-3)ppm/ degrees C, regardless of pH, these coefficients were highly pH-dependent for phosphatidic acid, phosphatidylethanolamine and phosphatidylserine, exhibiting maximal variations with the deprotonation of the headgroup, particularly for phosphatidic acid. These trends indicate the importance of H-bonding on delta and Deltadelta/DeltaT for phospholipid resonances.