Quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is used to determine changes in individual protein levels in complex protein mixtures. To provide reliable data, the software used for 2-D gel image analysis must provide a linear response over a wide dynamic range of data output. Here, we show that Phoretix 2D Full analysis of 2-D gels stained with colloidal Coomassie Brilliant Blue G-250 can provide a linear measure of changes in protein quantity. We show using a complex mixture of Arabidopsis thaliana proteins, that this is true for essentially all focused proteins, in a data output range greater than three orders of magnitude. An analysis of the factors that affect errors in the results demonstrated that reproducibility of the data is significantly improved by user seeding, whereas it is reduced by use of the background subtraction algorithms.