Background: The proteomics literature has seen a proliferation of publications that seek to apply the rapidly improving technology of 2D gels to study various biological systems. However, there is a dearth of systematic studies that have investigated appropriate statistical approaches to analyse the data from these experiments. Results: Comparison of the effects of statistical pre-processing on the results of two sample t-tests suggests that the results of 2D gel experiments and by extension the conclusions derived from these experiments are not independent of the statistical protocol used. Conclusions: This study suggests that there is a need for well-conducted validation studies to establish optimal statistical techniques to be used on such data sets. © 2005 Meleth et al; licensee BioMed Central Ltd.