The expression of housekeeping and/or immunoproteasomes in isolated thymic stroma subsets has so far not been analyzed but may have important consequences for self peptide repertoires presented by MHC class I molecules during positive and negative thymic selection. Here we determined the expression of housekeeping and immunoproteasome beta subunits and of PA28 in positively and negatively selecting stroma subsets. Positively selecting cortical thymic epithelial cells (cTEC) expressed only housekeeping but no immunoproteasome beta subunit mRNA and proteins. However, immunoproteasome beta subunits could be induced in cTEC by infection with Listeria monocytogenes or injection of IFN-gamma. In negatively selecting stroma including medullary epithelial cells and dendritic cells, incomplete and low representation of housekeeping beta subunit proteins but high and complete expression of immunoproteasome beta subunit proteins suggests absence of proper housekeeping proteasomes and predominance of immunoproteasomes. Expression of immunoproteasome beta subunits in negatively selecting stroma was independent of IFN-gamma receptor as shown in knockout (KO) mice. Absence of LMP2 altered thymic selection of the MHC class I-restricted transgenic P14 TCR in KO mice. The data suggest that negative selection may primarily involve immunoproteasome peptide repertoires and that peripheral infection may influence peptide repertoires involved in positive selection.