Background: Array-based comparative genomic hybridization (array-CGH) is a recently developed technique for analyzing changes in DNA copy number. As in all microarray analyses, normalization is required to correct for experimental artifacts while preserving the true biological signal. We investigated various sources of systematic variation in array-CGH data and identified two distinct types of spatial effect of no biological relevance as the predominant experimental artifacts: continuous spatial gradients and local spatial bias. Local spatial bias affects a large proportion of arrays, and has not previously been considered in array-CGH experiments. Results: We show that existing normalization techniques do not correct these spatial effects properly. We therefore developed an automatic method for the spatial normalization of arrayCGH data. This method makes it possible to delineate and to eliminate and/or correct areas affected by spatial bias. It is based on the combination of a spati...