We statistically analysed various factors to get accurate estimates of protein quantities from two-dimensional gels. Yeast proteins were labelled with (35)S or stained with Coomassie Brilliant Blue G-250, and spots were automatically quantified with software packages Kepler, ImageQuaNT, Melanie 3.0 and Progenesis. The different software packages proved to have very similar performances. With (35)S-labelled actin spot as a reference, we studied the staining efficiency of colloidal Coomassie blue as a function of amino acid composition of the protein, and derived an equation to estimate the number of molecules per cell from blue-stained proteins. Absolute quantification of most glycolytic enzymes was carried out in two yeast strains.