Objective: To identify differentially expressed genes in the mechanically unloaded rat heart by suppression subtractive hybridization. Methods: In male Wistar-Kyoto rats mechanical unloading was achieved by infrarenal heterotopic heart transplantation. Differentially expressed genes were investigated systematically by suppression subtractive hybridization. Selected targets were validated by northern blot analysis, Real-time RT-PCR and immunoblot analysis. Maximal ADP-stimulated oxygen consumption (state 3) was measured in isolated mitochondria. Results: Transplantation caused atrophy (heart-to-body weight-ratio: 1.6±0.1 vs. 2.4±0.1; p<0.001). We picked 1880 clones from the subtractive hybridization procedure (940/940: forward/reverse runs assessing up- or downregulation). The first screen verified 465/140, the second screen 67/30 clones. 24/23 clones were sequenced and 9/14 homologies to known genes were found. Specifically, we identified reduced mRNA expression of complex I (-49%, p<0.05) and II (-61%, p<0.001) of the respiratory chain. Significant reductions were also observed on the respiratory chain protein level (complex I: -42%, p<0.01; complex II: -57%, p<0.05; complex IV: -65%, p<0.05). Consistent with changes in gene and protein expression, state 3 respiration was significantly decreased in isolated mitochondria of atrophied hearts, both with glutamate and succinate as a substrate (natomsO/min/mg: glutamate 85±27 vs. 224±32, p<0.01; succinate 59±18 vs. 154±30, p<0.05). Conclusions: Subtractive hybridization indicates major changes in overall gene expression by mechanical unloading and specifically identified downregulation of respiratory chain genes. This observation is functionally relevant and provides a mechanism for the regulation of respiratory capacity in response to chronic mechanical unloading.