Limited data are available in humans regarding the molecular biology of hibernating myocardium (HM). The aim of this study was to identify gene expression patterns distinctive for human HM. We compared in patients with ischemic left ventricular dysfunction the gene expression profile of myocardial biopsies from HM (n = 5), as identified by positron emission tomography, with expression profiles of matched biopsies from normally perfused myocardium by using cDNA array analysis. Gene-specific polymerase chain reaction of selected genes and immunohistochemical staining of desmoplakin were used to validate our technical approach. Of 4171 transcripts examined, we identified 86 to be differentially expressed. Compared to normally perfused myocardium, 21 genes showed an increased expression and 65 genes a decreased expression in HM. Functional clustering revealed changes in the expression of genes associated with transcription, protein modification and phosphorylation, regulation of apoptosis, and intercellular communication. Besides the reported upregulation of beta-adrenergic receptor kinase-1 in heart failure, we observed new gene expression patterns, such as the upregulation of fas-activated serine/threonine kinase (FAST) or reduced expression of desmoplakin. Downregulation of desmoplakin in cardiomyocytes from HM was also seen on the protein level. Gene expression analysis provided novel insights into the pathophysiological changes of HM. Impaired intercellular communication as suggested by decreased expression of desmoplakin may be an important feature of contractile dysfunction in HM.