Abstract Motivation: Affymetrix GeneChips® are common 3′ profiling platforms for quantifying gene expression. Using publicly available datasets of expression profiles from human and mouse experiments, we sought to characterize features of GeneChip® data to better compare and evaluate analyses for differential expression, regulation and clustering. We uncovered an unexpected order dependence in expression data that holds across a variety of chips in both human and mouse data. Results: Order dependence among GeneChips® affected relative expression measures pre-processed and normalized with the Affymetrix MAS5.0 algorithm and the robust multi-array average summarization method. The effect strongly influenced detection calls and tests for differential expression and can potentially significantly bias experimental results based on GeneChip® profiling. Contact: [email protected] Supplementary information: Supplementary Material, including links to files of ordered transcripts and supporting analyses, is available at the authors' websites, at http://www-math.cudenver.edu/~kbjork/research/, and http://www-math.cudenver.edu/~kk/research/.