ABSTRACT Little is known regarding the biological roles of archaeal proteases. The haloarchaeon Haloferax volcanii is an ideal model for understanding these enzymes, as it is one of few archaea with an established genetic system. In this report, a series of H. volcanii mutant strains with markerless and/or conditional knockouts in each known proteasome gene was systematically generated and characterized. This included single and double knockouts of genes encoding the 20S core α1 ( psmA ), β ( psmB ), and α2 ( psmC ) subunits as well as genes ( panA and panB ) encoding proteasome-activating nucleotidase (PAN) proteins closely related to the regulatory particle triple-A ATPases (Rpt) of eukaryotic 26S proteasomes. Our results demonstrate that 20S proteasomes are required for growth. Although synthesis of 20S proteasomes containing either α1 or α2 could be separately abolished via gene knockout with little to no impact on growth, conditional depletion of either β alone or α1 and α2 together rendered the cells inviable. In contrast, the PAN proteins were not essential based on the robust growth of the panA panB double knockout strain. Deletion of genes encoding either α1 or PanA did, however, render cells more sensitive to growth on organic versus inorganic nitrogen sources and hypo-osmotic stress and limited growth in the presence of l -canavanine. Abolishment of α1 synthesis also had a severe impact on the ability of cells to withstand thermal stress. This contrasted with what was seen for panA knockouts, which displayed enhanced thermotolerance. Together, these results provide new and important insight into the biological role of proteasomes in archaea.