In the course of 50 S ribosome assembly in vitro, the L-proteins associate consecutively with the rRNA in two main groups. The first assembly group is found on the reconstitution intermediate particle RI50(1), the second group being added to form the RI50(2) particle. The analysis presented here allows for the first time all the ribosomal proteins to be assigned to one or the other of these groups. The following 22 proteins are found in the RI50(1) particle: L1, (L2), L3, L4, (L5), L7/12, L9, L10, L11, (L13), L15, L17, (L18), L20, L21, L22, L23, L24, (L26), L29, (L33), (L34). The assembly map has been arranged accordingly. The assembly interdependences of six proteins (L28, L30, L31, L32, L33, and L34) were assessed and that of L14 revised by means of a large number of assembly mapping experiments, the final decisive steps of which are documented here. The interactions of these latter seven proteins and the incorporation of five additional assembly interdependences are integrated into the 50 S assembly map, which now contains all the components of the 50 S subunit, with the exception of protein L26; this protein is known to be the same as protein S20 and is predominantly associated with the 30 S subunit.