Brief periods of ischemia can protect the heart from a subsequent longer coronary artery occlusion, the phenomenon called ischemic preconditioning. The mechanism of ischemic preconditioning are not well understood. A number of genes have been shown to play roles in cytoprotective effect of ishemic preconditioning, but there are clearly many missing elements to be found. Suppression subtractive hybridization was used to construct cDNA libraries enriched for genes up-regulated during ischemic preconditiong. After being confirmed by reverse Northern dot blot for differential expression, the selected genes were sequenced and searched in GenBank for homology analysis. Many nuclear-encoded genes that were up-regulated during ischemic preconditioning participate in cytoprotection. The 18 novel ESTs were banked into GenBank with accession numbers. The specificity of this response was confirmed by RT-PCR and Northern blot. Understanding the genes up-regulated during ischemic preconditioning may open new avenues for therapy in ischemic heart disease.