(31)P NMR spectroscopy is known to be a fast and accurate method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantitated separately in (31)P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. For solvent systems resulting in the formation of two phases, the effects of varying water and methanol content on chemical shift and line width of phospholipid signals have been previously determined. However, little attention has been paid to the influence that other extract components may exert on signal separation. We present, for the first time, a systematic and comprehensive study of (31)P NMR chemical shift as a function of four experimental parameters: (i) extract concentration, (ii) concentration of chelating agent, (iii) pH value of the aqueous component of the solvent system, and (iv) temperature of the NMR measurement. This multiparametric study provides methodological guidelines for predictable and reproducible manipulation of (31)P NMR spectra of brain phospholipids. It also provides a database for rational and efficient optimization of phospholipid spectra from other body tissues, cultured cells, and phospholipid-containing biofluids.