The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200-, 500-, and, 1000-fold for proteins analyzed by SDS-PAGE. SYPRO Ruby protein gel stain was also evaluated in two-dimensional electrophoretic (2-DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 nug total protein across gels. In addition to validating the 2-DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio-Rad). In each program, we analyzed the 2-DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel-to-gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000-fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.