The ubiquitin-proteasome pathway, which degrades intracellular proteins, is involved in numerous cellular processes, including the supply of immunocompetent peptides to the antigen presenting machinery. Proteolysis by proteasomes is conducted by three β subunits, β1, β2, and β5, of the 20S proteasome. Recently, a novel β subunit expressed exclusively in cortical thymic epithelial cells was discovered in mice. This subunit, designated β5t, is a component of the thymoproteasome, a specialized type of proteasomes implicated in thymic positive selection. In this study, we show that, like its mouse counterpart, human β5t is expressed exclusively in the thymic cortex. Human β5t was expressed in approximately 80% of cortical thymic epithelial cells and some cortical dendritic cells. Human β5t was incorporated into proteasomes with two other catalytically active β subunits β1i and β2i, forming 20S proteasomes with subunit compositions characteristic of thymoproteasomes. The present study demonstrates, for the first time, the existence of thymoproteasomes in the human thymic cortex, indicating that thymoproteasome function is likely conserved between humans and mice.