Microarray data must be normalized because they suffer from multiple biases. We have identified a source of spatial experimental variability that significantly affects data obtained with Cy3/Cy5 spotted glass arrays. It yields a periodic pattern altering both signal (Cy3/Cy5 ratio) and intensity across the array. Using the variogram, a geostatistical tool, we characterized the observed variability, called here the spotting effect because it most probably arises during steps in the array printing procedure. The spotting effect is not appropriately corrected by current normalization methods, even by those addressing spatial variability. Importantly, the spotting effect may alter differential and clustering analysis.