DNA microarrays represent a powerful technology whose use has been hampered by the uncertainty of whether the same principles, established on a scale typical for membrane hybridizations, apply when using the smaller, rigid support of microarrays. Our goal was to understand how the number and position of base pair mismatches, probe length and their G+C content affect the intensity and specificity of the hybridization signal. One set of oligonucleotides (50-mers) based on three regions of the Bacillus thuringiensis cry1Aa1 gene possessing 30%, 42%, and 56% G+C content, a second set with similar G+C content (37% to 40%) but different lengths (30 to 100 bases), and finally amplicon probes (101 to 3000 base pairs) with G+C contents of 37% to 39%, were used. Probes with mismatches distributed over their entire length were the most specific, while those with mismatches grouped at either the 3? or 5?-end were the least specific. Hybridizations done at 8 to 13 �C below the calculated Tm of perfectly matched probes, as compared to the widely used lower temperatures of 20 to 25 �C, enhanced probe discrimination. Longer probes produced higher fluorescent hybridization signals than shorter ones. These results should help to optimize the design of oligonucleotide-based DNA microarrays.