We present evidence of a potentially serious source of error intrinsic to all spotted cDNA microarrays that use I.M.A.GE. clones of expressed sequence tags (ESTs). We found that a high proportion of these EST sequences contain 5' end poly(dT) sequences that are remnants from the oligo (dT)-primed reverse transcription of polyadenylated mRNA templates used to generate EST cDNA for sequence clone libraries. Analysis of expression data from two single-dye cDNA microarray experiments showed that ESTs whose sequences contain repeats of consecutive 5'-end dT residues appeared to be strongly co-expressed, while expression data of all other sequences exhibited no such pattern. Our anlaysis suggests that expression data from sequences containing 5' poly(dT) tracts is more likely to be due to systematic cross-hybridization of these poly(dT) tracts than true mRNA co-expression. This indicates that existing data generated by cDNA microarrays containing I.M.A.G.E. clone ESTs should be filtered to remove expression data containing significant 5' poly(dT) tracts.