Dilated cardiomyopathy is now the leading cause of cardiovascular morbidity and mortality. While the molecular basis of this disease remains uncertain, evidence is emerging that gene expression profiles of left ventricular myocardium isolated from failing versus nonfailing patients differ dramatically. In this study, we use high-density oligonucleotide microarrays with ∼22,000 probes to characterize differences in the expression profiles further. To facilitate interpretation of experimental data, we evaluate algorithms for normalization of hybridization data and for computation of gene expression indices using a control spike-in data set. We then use these methods to identify statistically significant changes in the expression levels of genes not previously implicated in the molecular phenotype of heart failure. These regulated genes take part in diverse cellular processes, including transcription, apoptosis, sarcomeric and cytoskeletal function, remodeling of the extracellular matrix, membrane transport, and metabolism.