AbstractThe proteasome core particle (CP) is a cytoplasmic and nuclear protease complex and is comprised of two α‐rings and two β‐rings stacked in order of αββα. The assembly of CP proceeds by ordered recruitment of β‐subunits on an α‐ring with help of assembly chaperones PAC1‐PAC2, PAC3‐PAC4, and UMP1. However, the mechanism of α‐ring formation remains unsolved. Here, we show that α4, α5, α6, and α7 form a core intermediate as the initial process of α‐ring assembly, which requires PAC3‐PAC4. α1 and α3 can be incorporated independently into the core α4–α7 intermediate, whereas α2 incorporation is dependent on preceding incorporation of α1. Through these processes, PAC1‐PAC2 prevents nonproductive dimerization of α‐ring assembly intermediates. We also found that PAC1‐PAC2 overrides the effect of nuclear localization signals of α‐subunits and retains α‐ring assembly intermediates in the cytoplasm. Our results first show a detailed assembly pathway of proteasomal α‐ring and explain the mechanism by which CP assembly occurs in the cytoplasm.