The accuracy and precision of the quantification of metabolite concentrations in in vivo 1H NMR spectroscopy are affected by linewidth and signal-to-noise ratio (SNR). To study the effect of both factors in in vivo 1H NMR spectra acquired at ultrahigh field, a reference spectrum was generated by summing nine in vivo 1H NMR spectra obtained in rat brain with a STEAM sequence at 16.4 T. By progressive deterioration of linewidth and SNR, 6400 single spectra were generated. In an accuracy study, the variation in the mean concentrations of five metabolites was mainly dependent on SNR, whereas 11 metabolites were predominantly susceptible to the linewidth. However, the standard deviations of the concentrations obtained were dependent almost exclusively on the SNR. An insignificant correlation was found between most of the heavily overlapping metabolite peaks, indicating independent and reliable quantification. Two different approaches for the consideration of macromolecular signals were evaluated. The use of prior knowledge derived by parameterization of a metabolite-nulled spectrum demonstrated improved fitting quality, with reduced Cramér–Rao lower bounds, compared to the calculation of a regularized spline baseline. Copyright © 2012 John Wiley & Sons, Ltd.