The 26S double-capped proteasome is assembled in a hierarchic event that is orchestrated by dedicated set of chaperons. To date, all stoichiometric subunits are considered to be present in equal ratios, thus providing symmetry to the double-capped complex. Here, we show that although the vast majority (if not all) of the double-capped 26S proteasomes, both 19S complexes, contain the ubiquitin receptor Rpn10/S5a, only one of these 19S particles contains the additional ubiquitin receptor Rpn13, thereby defining asymmetry in the 26S proteasome. These results were validated in yeast and mammals, utilizing biochemical and unbiased AQUA-MS methodologies. Thus, the double-capped 26S proteasomes are asymmetric in their polyubiquitin binding capacity. Our data point to a potential new role for ubiquitin receptors as directionality factors that may participate in the prevention of simultaneous substrates translocation into the 20S from both 19S caps.