Quantification of localized in vivo brain 1H spectra is in general very difficult due to excessive spectral overlap. In addition, intensity distortions may result from the effects of the NMR pulse sequence on the spins. This paper describes an approach to solving these problems. It comprises optimization of the pulse sequence; correction of the experimental lineshape; determination of intensity distortions, of relative line positions, and of linewidths using model solutions; and incorporation of the thus obtained prior knowledge into a nonlinear least-squares spectral fitting procedure. This approach resulted in greatly improved accuracy, precision, and reliability of the quantitation of our in vivo spectra of rat brain, and enabled us to estimate absolute metabolite concentrations.