To profile gene expression patterns involved in the direct myocardial effect of cholesterol-enriched diet-induced hyperlipidemia, we monitored global gene expression changes by DNA microarray analysis of 3200 genes in rat hearts. Twenty-six genes exhibited significant up-regulation and 25 showed down-regulation in hearts of rats fed a 2% cholesterol-enriched diet for 8 weeks as compared to age-matched controls. The expression changes of 12 selected genes were also assessed by real-time quantitative polymerase chain reaction. Genes with altered expression in the heart due to hyperlipidemia included procollagen type III, cofilin/destrin, tensin, transcription repressor p66, synaptic vesicle protein 2B, Hsp86, chaperonin subunit 5epsilon, metallothionein, glutathione S-transferase, protein kinase C inhibitor, ATP synthase subunit c, creatine kinase, chloride intracellular channel 4, NADH oxidoreductase and dehydrogenase, fibronectin receptor beta chain, CD81 antigen, farnesyltransferase, calreticulin, disintegrin, p120 catenin, Smad7, etc. Although some of these genes have been suspected to be related to cardiovascular diseases, none of the genes has been previously shown to be involved in the mechanism of the cardiac effect of hyperlipidemia.