The importance of proteasomes in governing the intracellular protein degradation process has been increasingly recognized. Recent investigations indicate that proteasome complexes may exist in a species- and cell-type-specific fashion. To date, despite evidence linking impaired protein degradation to cardiac disease phenotypes, virtually nothing is known regarding the molecular composition, function, or regulation of cardiac proteasomes. We have taken a functional proteomic approach to characterize 26S proteasomes in the murine heart. Multidimensional chromatography was used to obtain highly purified and functionally viable cardiac 20S and 19S proteasome complexes, which were subjected to electrophoresis and tandem mass spectrometry analyses. Our data revealed complex molecular organization of cardiac 26S proteasomes, some of which are similar to what were reported in yeast, whereas others exhibit contrasting features that have not been previously identified in other species or cell types. At least 36 distinct subunits (17 of 20S and 19 of 19S) are coexpressed and assembled as 26S proteasomes in this vital cardiac organelle, whereas the expression of PA200 and 11S subunits were detected with limited participation in the 26S complexes. The 19S subunits included a new alternatively spliced isoform of Rpn10 (Rpn10b) along with its primary isoform (Rpn10a). Immunoblotting and immunocytochemistry verified the expression of key alpha and beta subunits in cardiomyocytes. The expression of 14 constitutive alpha and beta subunits in parallel with their three inducible subunits (beta1i, beta2i, and beta5i) in the normal heart was not expected; these findings represent a distinct level of structural complexity of cardiac proteasomes, significantly different from that of yeast and human erythrocytes. Furthermore, liquid chromatography/tandem mass spectroscopy characterized 3 distinct types of post-translational modifications including (1) N-terminal acetylation of 19S subunits (Rpn1, Rpn5, Rpn6, Rpt3, and Rpt6) and 20S subunits (alpha2, alpha5, alpha7, beta3, and beta4); (2) N-terminal myristoylation of a 19S subunit (Rpt2); and (3) phosphorylation of 20S subunits (eg, alpha7)). Taken together, this report presents the first comprehensive characterization of cardiac 26S proteasomes, providing critical structural and proteomic information fundamental to our future understanding of this essential protein degradation system in the normal and diseased myocardium.