Toxicogenomics is an emerging field combining genomics and bioinformatics to identify and characterize mechanisms of toxicity of compounds. One of the main tools used in toxicogenomics is DNA microarrays. We have used a novel strategy to create a library highly enriched for genes expressed in the liver under hepatotoxic conditions. Using this library, we have created a new oligonucleotide microarray dedicated to the study of rat liver function. Oligonucleotide probes for these genes were designed and used in experimental hybridization studies to deduce the correct sequence orientation and to determine those sequences exhibiting differential regulation under a variety of toxicity-related treatments and conditions. The final array was benchmarked on treatments with 3-methylcholanthrene, Aroclor 1254, and cyclopropane carboxylic acid. Our results showed that the subtractive hybridization greatly enriched for genes regulated during a hepatotoxic response. Overall, our strategy for design of a new rat toxicology microarray can be applied to other systems as well and should aid greatly in the development of microarrays targeted for specific scientific areas.