Gene expression analysis using customized or focused DNA microarrays is favorable because of a reduction in the cost and time needed for the analysis. To examine the effect of chemicals on the liver, we developed an in-house cDNA microarray system, mouse Liver Stress Array ver. 1.0, containing 355 unique genes involved in drug metabolism, inflammation and liver-related proteins. These genes were selected for sensing the homeostasis of the liver and based on the information of liver transcriptome revealed by serial analysis of gene expression. By using this customized microarray, we analyzed gene expression changes in the mouse liver treated by 11 known hepatotoxicants. Gene expression measurements corresponding to the in vivo response to known hepatotoxicants revealed that profiles of chemicals with similar mechanisms clustered together. For each of the chemicals tested, several genes that were induced or repressed were common in each chemical exposure, whereas other genes were unique for the specific class compound. Ingenuity pathways analysis revealed that significant alterations in gene expression occurred in a number of biological networks by these treatments. Although the genes spotted on our array was limited to a highly focused set for toxicity classification, this work provides proof of concept that patterns of gene regulation assessed by a focused array system are useful to classify unknown chemicals.