The effect of different amplification and labeling methods on DNA microarray expression results has not been previously delineated. To analyze the variation associated with widely accepted T7-based RNA amplificationand labeling methods, aliquots of the Stratagene Human Universal Reference RNA were labeled using three eukaryotic target preparation methods followed by uniform replicate array hybridization (Affymetrix U95Av2). Method-dependent variability was observed in the yield and size distribution of labeled products, as well as in the gene expression results. A significant increase in short transcripts, when compared to unamplified mRNA, was observed in methods with long in vitro transcription reactions. Intramethod reproducibility showed correlation coefficients >0.99, whereas intermethod comparisons showed coefficients ranging from 0.94 to 0.98 and a nearly twofold increase in coefficient of variation. Fold amplification for each method positively correlated with the number of genes present. Our experiments uncovered two factors that introduced significant bias in gene expression data: the number of labeled nucleotides, which introduces sequence-dependent bias, and the length of the in vitro transcription reaction, which introduces transcript size-dependent bias. This study provides evidence that variability in expression data may be caused, in part, by differences in amplification and labeling protocols.