Further studies were made on the reassembly of 50S ribosomal subunits from proteins and RNAs of E. coli. The reassembled particles had high activity in poly U-directed polyphenylalanine synthesis and their sucrose sedimentation properties were similar to those of the original intact particles. Several factors affecting the reassembly were examined. The optimal pH for solubilization of ribosomal proteins was pH 9.5, and the optimal Tris concentration was 0.75 to 1.00 M. In the reassembly mixture the pH was adjusted to 8.2 A sharp optimum magnesium ion concentration of 6 to 10 mM was observed. The reassembly required 0.2 to 0.5 M KCl, the optimum concentration being 0.40 M. On incubation for 20 min a temperature of 34 and 40 degrees was necessary, 37 degrees being best. Oligonucleotides, which we previously added to the reassembly mixture were found not to be necessary for inhibition of RNase II [EC 3.1.4.20] activity remaining in the reaction was found necessary to dialyze the reassembly mixture against a buffer containing 10 mM magnesium ion after the incubation. Simultaneous reassembly of 30 and 50S subunits with time was observed, showing that 70S ribosomes were formed first and that they then dissociated into subunits. Reassembly of 50S subunits from their component proteins and RNAs was completely dependent on either 30S particles or the simultaneous reassembly of 30S subunits. Other critical factors affecting the reassembly of 50S subunits must be examined, since the reproducibility of this reassembly is only about 60%, even under the above controlled conditions.