The quality of NMR spectra in general and of spectra to be used for analysis of compound mixtures in particular is essentially defined by two basic parameters: signal-to-noise ratio and spectral resolution. The latter is determined by signal dispersion (chemical shift differences) and line widths. The present study focuses on multiparametric optimization of spectral resolution in (31)P NMR spectra of phospholipids from brain tissue extracts. This report presents, for the first time, a systematic and comprehensive study of phospholipid (31)P NMR line widths as a function of four experimental parameters: (i) extract concentration, (ii) concentration of a chelating agent, (iii) pH of the aqueous component of the solvent system, and (iv) temperature of the NMR measurement. Theoretical underpinnings of observed line width variations (transversal relaxation effects) are briefly discussed. In conjunction with an analogous, concurrently published report on chemical shift effects in the same tissue extract system, this multiparametric line width study provides a complete set of methodological guidelines for (i) generating well-defined tissue extracts, and (ii) choosing matched and optimized measurement conditions for highly reproducible and well-resolved (31)P NMR spectra of brain phospholipids. This study also offers a comprehensive database and a strategy for rational and efficient optimization of phospholipid spectra from other tissue extracts.