Updated project metadata. Ribosomes, macromolecular machines producing a cell´s protein content, are formed from their RNA and protein components in a dynamic process referred to as ribosome assembly. While ribosome assembly in the cell starts with the successive synthesis of ribosomal RNA (rRNA) by the RNA polymerase holoenzyme, and requires numerous assembly factors, (Kaczanowska & Ryden-Aulin, 2007; Shajani et al., 2011), the process can be accomplished in vitro, using purified ribosomal components and scalable reaction conditions (Nierhaus & Dohme, 1974; Traub & Nomura, 1968). To obtain structural and conceptual insights in the early phase of the process, we performed the in vitro assembly reaction of the bacterial 50S subunit as a time course reaction, analyzed samples by sucrose density gradient ultracentrifugation, activity assay, quantitative mass spectrometry (qMS) and cryo-electron microscopy (cryo-EM). Our structural analysis reveals that early 50 assembly initiates with 23S rRNA domain I and occurs in a domain-wise fashion, while late 50S assembly proceeds incrementally. Notably, in both phases parallel pathways are utilized. Furthermore, we find that both ribosomal proteins and folded rRNA helices, occupying surface exposed regions on pre-50S particles, induce, or stabilize rRNA folds within adjacent regions, thereby creating cooperativity. Our study provides new insights into fundamental principles governing ribosome assembly.