Absolute metabolite concentrations have been estimated for nucleoside triphosphate and P(i) from in vivo 31P MR measurements using ISIS localization in a rat tumour model, and the results have been compared to those obtained from acid extracts of the tumours. The aim of the experiment was to assess the performance of four different spectral analysis techniques used for absolute quantitation. The spectral analysis techniques used were two frequency domain methods (peak area integration and Lorentzian fitting--FITSPEC) and two time domain methods (VARPRO and HLSVD). The spectra were acquired in blocks so that the degradation in performance of the four spectral analysis methods with decreasing signal-to-noise ratio (SNR) could be compared and referenced. This and the inclusion of a sophisticated method incorporating prior knowledge yields a more realistic and comprehensive protocol than previously published comparisons. The results suggest that VARPRO is the method of choice for quantitative analysis of tumour 31P MR spectra, giving the most reliable results at low SNR.