The proteasome-dependent protein degradation participates in multiple essential cellular processes. Modulation of proteasomal activities may alter cardiac function and disease phenotypes. However, cardiovascular studies reported thus far, yielded conflicting results. We hypothesized that a contributing factor to the contradicting literature may be caused by existing proteasome heterogeneity in the myocardium. In this investigation, we provide the very first direct demonstration of distinct proteasome subpopulations in murine hearts. The cardiac proteasome subpopulations differ in their molecular compositions and proteolytic activities. Furthermore, they were distinguished from proteasome subpopulations identified in murine livers. The study was facilitated by the development of novel protocols for in-solution isoelectric focusing of multi-protein complexes in a laminar flow, which support an average resolution of 0.04 pH units. Utilizing these protocols, the majority of cardiac proteasome complexes displayed an isoelectric point of 5.26, with additional subpopulations focusing in the range from pH 5.10-5.33. In contrast, the majority of hepatic 20S had a pI of 5.05 and focused from pH 5.01-5.29. Importantly, proteasome subpopulations degraded specific model peptides with different turnover rates. Among cardiac subpopulations, proteasomes with an approximate pI of 5.21 showed 40% higher trypsin-like activity than those with pI 5.28. Distinct proteasome assembly may be a contributing factor to variations in proteolytic activities, since proteasomes with pI 5.21 contained 58% less of the inducible subunit ß2i compared to those with pI 5.28. In addition, dephosphorylation of 20S demonstrated that besides molecular composition, posttranslational modifications largely contribute to its pI. These data suggest the possibility of mixed 20S proteasome assembly, a departure from the currently hypothesized two subpopulations: constitutive and immuno-forms. The identification of multiple distinct proteasome subpopulations in heart provides key mechanistic insights for achieving selective and targeted regulation of this essential protein degradation machinery. Thus, proteasome subpopulations may serve as novel therapeutic targets in the myocardium.