Proteasomes provide the main route of intracellular protein degradation. They consist of a central protease, termed the 20S proteasome, or core particle (CP), that partners with one or more regulatory complexes. The quaternary structure of the CP is conserved across all domains of life and is comprised of four coaxially stacked heptameric rings formed by structurally related alpha and beta subunits. In eukaryotes, biogenesis of the CP is generally assumed to involve the obligate formation of alpha-rings. These serve as templates upon which beta subunits assemble to form half-proteasomes which dimerize to give rise to CP. Here, we demonstrate the in vivo existence of an assembly-competent intermediate containing an incomplete set of both alpha and beta subunits. The novel intermediate exhibits a precursor-product relationship with the well characterized CP assembly intermediate, the 13S. This is the first evidence that eukaryotic CP, like its archaeal and bacterial counterparts, can assemble in an alpha-ring independent manner. (C) 2020 Elsevier Inc. All rights reserved.