Using Escherichia coli cDNA microarray slides and Affymetrix GeneChips, we study how the relative position of probes on microarrays affects the cross-correlation of gene expression profiles. We find that in cDNA arrays, every spot located within the same block is affected by a similar, experiment-specific bias. As a result, the cross-correlation between some gene expression profiles is significantly altered, depending on the similarity between these “block-dependent” biases through the series of cDNA microarray experiments. In addition, the position of probes within the blocks can also contribute to the measured gene expression. We outline the necessary steps to computationally identify and correct these biases.